Genetic products differentially expressed in tumors and the use thereof

ABSTRACT

The invention relates to the identification of genetic products expressed in association with tumors and to coding nucleic acids for the expressed products. An embodiment of the invention also relates to the therapy and diagnosis of disease in which the genetic products are aberrantly expressed in association with tumors, proteins, polypeptides and peptides which are expressed in association with tumors, and to the nucleic acids coding for the polypeptides, peptides and proteins.

This application is a United States national stage filing under 35U.S.C. §371 of international (PCT) application No. PCT/EP2003/013091,filed Nov. 21, 2003, and designating the US, which claims priority toGerman application no. 102 54 601.0 filed Nov. 22, 2002.

BACKGROUND OF THE INVENTION

Despite interdisciplinary approaches and exhaustive use of classicaltherapeutic procedures, cancers are still among the leading causes ofdeath. More recent therapeutic concepts aim at incorporating thepatient's immune system into the overall therapeutic concept by usingrecombinant tumor vaccines and other specific measures such as antibodytherapy. A prerequisite for the success of such a strategy is therecognition of tumor-specific or tumor-associated antigens or epitopesby the patient's immune system whose effector functions are to beinterventionally enhanced. Tumor cells biologically differ substantiallyfrom their nonmalignant cells of origin. These differences are due togenetic alterations acquired during tumor development and result, interalia, also in the formation of qualitatively or quantitatively alteredmolecular structures in the cancer cells. Tumor-associated structures ofthis kind which are recognized by the specific immune system of thetumor-harboring host are referred to as tumor-associated antigens. Thespecific recognition of tumor-associated antigens involves cellular andhumoral mechanisms which are two functionally interconnected units: CD4⁺and CD8⁺ T lymphocytes recognize the processed antigens presented on themolecules of the MHC (major histocompatibility complex) classes II andI, respectively, while B lymphocytes produce circulating antibodymolecules which bind directly to unprocessed antigens. The potentialclinical-therapeutical importance of tumor-associated antigens resultsfrom the fact that the recognition of antigens on neoplastic cells bythe immune system leads to the initiation of cytotoxic effectormechanisms and, in the presence of T helper cells, can cause eliminationof the cancer cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, acentral aim of tumor immunology is to molecularly define thesestructures. The molecular nature of these antigens has been enigmaticfor a long time. Only after development of appropriate cloningtechniques has it been possible to screen cDNA expression libraries oftumors systematically for tumor-associated antigens by analyzing thetarget structures of cytotoxic T lymphocytes (CTL) (van der Bruggen etal., Science 254:1643-7, 1991) or by using circulating autoantibodies(Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To thisend, cDNA expression libraries were prepared from fresh tumor tissue andrecombinantly expressed as proteins in suitable systems. Immunoeffectorsisolated from patients, namely CTL clones with tumor-specific lysispatterns, or circulating autoantibodies were utilized for cloning therespective antigens.

In recent years a multiplicity of antigens have been defined in variousneoplasias by these approaches. However, the probes utilized for antigenidentification in the classical methods illustrated above areimmunoeffectors (circulating autoantibodies or CTL clones) from patientsusually having already advanced cancer. A number of data indicate thattumors can lead, for example, to tolerization and anergization of Tcells and that, during the course of the disease, especially thosespecificities which could cause effective immune recognition are lostfrom the immunoeffector repertoire. Current patient studies have not yetproduced any solid evidence of a real action of the previously found andutilized tumor-associated antigens. Accordingly, it cannot be ruled outthat proteins evoking spontaneous immune responses are the wrong targetstructures.

BRIEF SUMMARY OF THE INVENTION

It was the object of the present invention to provide target structuresfor a diagnosis and therapy of cancers.

According to the invention, this object is achieved by the subjectmatter of the claims.

According to the invention, a strategy for identifying and providingantigens expressed in association with a tumor and the nucleic acidscoding therefor was pursued. This strategy is based on the fact thatparticular genes which are expressed in an organ specific manner, e.g.exclusively in colon, lung or kidney tissue, are reactivated also intumor cells of the respective organs and moreover in tumor cells ofother tissues in an ectopic and forbidden manner. First, data miningproduces a list as complete as possible of all known organ-specificgenes which are then evaluated for their aberrant activation indifferent tumors by expression analyses by means of specific RT-PCR.Data mining is a known method of identifying tumor-associated genes. Inthe conventional strategies, however, transcriptoms of normal tissuelibraries are usually subtracted electronically from tumor tissuelibraries, with the assumption that the remaining genes aretumor-specific (Schmitt et al., Nucleic Acids Res. 27:4251-60, 1999;Vasmatzis et al., Proc. Natl. Acad. Sci. USA. 95:300-4, 1998; Scheurleet al., Cancer Res. 60:4037-43, 2000).

The concept of the invention, which has proved much more successful,however, is based on utilizing data mining for electronically extractingall organ-specific genes and then evaluating said genes for expressionin tumors.

The invention thus relates in one aspect to a strategy for identifyingtissue-specific genes differentially expressed in tumors. Said strategycombines data mining of public sequence libraries (“in silico”) withsubsequent evaluating laboratory-experimental (“wet bench”) studies.

According to the invention, a combined strategy based on two differentbioinformatic scripts enabled new tumor genes to be identified. Thesehave previously been classified as being purely organ-specific. Thefinding that these genes are aberrantly activated in tumor cells allowsthem to be assigned a substantially new quality with functionalimplications. According to the invention, these tumor-associated genesand the genetic products encoded thereby were identified and providedindependently of an immunogenic action.

The tumor-associated antigens identified according to the invention havean amino acid sequence encoded by a nucleic acid which is selected fromthe group consisting of (a) a nucleic acid which comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 1-8,41-44, 51-59, 84, 117, and 119, a part or derivative thereof, (b) anucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c). In a preferredembodiment, a tumor-associated antigen identified according to theinvention has an amino acid sequence encoded by a nucleic acid which isselected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84,117, and 119. In a further preferred embodiment, a tumor-associatedantigen identified according to the invention comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 9-19, 45-48,60-66, 85, 90-97, 100-102, 105, 106, 111-116, 118, 120, 123, 124, and135-137, a part or derivative thereof.

The present invention generally relates to the use of tumor-associatedantigens identified according to the invention or of parts orderivatives thereof, of nucleic acids coding therefor or of nucleicacids directed against said coding nucleic acids or of antibodiesdirected against the tumor-associated antigens identified according tothe invention or parts or derivatives thereof for therapy and diagnosis.This utilization may relate to individual but also to combinations oftwo or more of these antigens, functional fragments, nucleic acids,antibodies, etc., in one embodiment also in combination with othertumor-associated genes and antigens for diagnosis, therapy and progresscontrol.

Preferred diseases for a therapy and/or diagnosis are those in which oneor more of the tumor-associated antigens identified according to theinvention are selectively expressed or abnormally expressed.

The invention also relates to nucleic acids and genetic products whichare expressed in association with a tumor cell.

Furthermore, the invention relates to genetic products, i.e. nucleicacids and proteins or peptides, which are produced by altered splicing(splice variants) of known genes or altered translation usingalternative open reading frames. In this aspect the invention relates tonucleic acids which comprise a nucleic acid sequence selected from thegroup consisting of sequences according to SEQ ID NOs: 3-5 of thesequence listing. Moreover, in this aspect, the invetion relates toproteins or peptides which comprise an amino acid sequence selected fromthe group consisting of the sequences according to SEQ ID NOs: 10 and12-14 of the sequence listing. The splice variants of the invention canbe used according to the invention as targets for diagnosis and therapyof tumor diseases.

In particular, the invention relates to the amino acid sequenceaccording to SEQ ID NO: 10 of the sequence listing which is encoded byan alternative open reading frame identified according to the inventionand differs from the previously described protein sequence (SEQ ID NO:9) in additional 85 amino acids at the N terminus of the protein.

Very different mechanisms may cause splice variants to be produced, forexample

-   -   utilization of variable transcription initiation sites    -   utilization of additional exons    -   complete or incomplete splicing out of single or two or more        exons,    -   splice regulator sequences altered via mutation (deletion or        generation of new donor/acceptor sequences),    -   incomplete elimination of intron sequences.

Altered splicing of a gene results in an altered transcript sequence(splice variant). Translation of a splice variant in the region of itsaltered sequence results in an altered protein which may be distinctlydifferent in the structure and function from the original protein.Tumor-associated splice variants may produce tumor-associatedtranscripts and tumor-associated proteins/antigens. These may beutilized as molecular markers both for detecting tumor cells and fortherapeutic targeting of tumors. Detection of tumor cells, for examplein blood, serum, bone marrow, sputum, bronchial lavage, bodilysecretions and tissue biopsies, may be carried out according to theinvention, for example, after extraction of nucleic acids by PCRamplification with splice variant-specific oligonucleotides. Inparticular, pairs of primers are suitable as oligonucleotides at leastone of which binds to the region of the splice variant which istumor-associated under stringent conditions. According to the invention,oligonucleotides described for this purpose in the examples aresuitable, in particular oligonucleotides which have or comprise asequence selected from SEQ ID NOs: 34-36, 39, 40, and 107-110 of thesequence listing. According to the invention, all sequence-dependentdetection systems are suitable for detection. These are, apart from PCR,for example gene chip/microarray systems, Northern blot, RNAseprotection assays (RDA) and others. All detection systems have in commonthat detection is based on a specific hybridization with at least onesplice variant-specific nucleic acid sequence. However, tumor cells mayalso be detected according to the invention by antibodies whichrecognize a specific epitope encoded by the splice variant. Saidantibodies may be prepared by using for immunization peptides which arespecific for said splice variant. In this aspect, the invention relates,in particular, to peptides which have or comprise a sequence selectedfrom SEQ ID NOs: 17-19, 111-115, 120, and 137 of the sequence listingand specific antibodies which are directed thereto. Suitable forimmunization are particularly the amino acids whose epitopes aredistinctly different from the variant(s) of the genetic product, whichis (are) preferably produced in healthy cells. Detection of the tumorcells with antibodies may be carried out here on a sample isolated fromthe patient or as imaging with intravenously administered antibodies. Inaddition to diagnostic usability, splice variants having new or alteredepitopes are attractive targets for immunotherapy. The epitopes of theinvention may be utilized for targeting therapeutically activemonoclonal antibodies or T lymphocytes. In passive immunotherapy,antibodies or T lymphocytes which recognize splice variant-specificepitopes are adaptively transferred here. As in the case of otherantigens, antibodies may be generated also by using standardtechnologies (immunization of animals, panning strategies for isolationof recombinant antibodies) with utilization of polypeptides whichinclude these epitopes. Alternatively, it is possible to utilize forimmunization nucleic acids coding for oligo- or polypeptides whichcontain said epitopes. Various techniques for in vitro or in vivogeneration of epitope-specific T lymphocytes are known and have beendescribed in detail (for example Kessler J H, et al. 2001, Sahin et al.,1997) and are likewise based on utilizing oligo- or polypeptides whichcontain the splice variant-specific epitopes or nucleic acids coding forsaid oligo- or polypeptides. Oligo- or polypeptides which contain thesplice variant-specific epitopes or nucleic acids coding for saidpolypeptides may also be used as pharmaceutically active substances inactive immunotherapy (vaccination, vaccine therapy).

The present invention also describes proteins which differ in nature anddegree of their secondary modifications in normal and tumor tissue (forexample Durand & Seta, 2000; Clin. Chem. 46: 795-805; Hakomori, 1996;Cancer Res. 56: 5309-18).

The analysis of protein modifications can be done in Western blots. Inparticular, glycosylations which as a rule have a size of several kDaresult in a higher overall mass of the target protein which can beseparated in an SDS-PAGE. For the detection of specific O- andN-glycosidic bonds protein lysates are incubated with O- orN-glycosylases (according to the instructions of the respectivemanufactures, for example, PNgase, endoglycosidase F, endoglycosidase H,Roche Diagnostics) prior to denaturation using SDS. Thereafter, aWestern blot is performed. If the size of target protein is reduced aspecific glycosylation can be detected in this manner followingincubation with a glycosidase and thus, also the tumor specificity of amodification can be analyzed. Protein regions which are differentiallyglycosylated in tumor cells and healthy cells are of particularinterest. Such differences in glycosylation, however, have hitherto onlybeen described for a few cell surface proteins (for example, Muc1).

According to the invention, it was possible to detect a differentialglycosylation for Claudin-18 in tumors. Gastrointestinal carcinomas,pancreas carcinomas, esophagus tumors, prostate tumors as well as lungtumors have a form of Claudin-18 which is glycosylated at a lower level.Glycosylation in healthy tissues masks protein epitopes of Claudin-18which are not covered on tumor cells due to lacking glycosylation.Correspondingly it is possible according to the invention to selectligands and antibodies which bind to these domains. Such ligands andantibodies according to the invention do not bind to Claudin-18 onhealthy cells since here the epitops are covered due to glycosylation.

As has been described above for protein epitopes which are derived fromtumor-associated splice variants it is thus possible to use thedifferential glycosylation to distinguish normal cells and tumor cellswith diagnostic as well as therapeutic intention.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which recognizes the tumor-associated antigenidentified according to the invention and which is preferably selectivefor cells which have expression or abnormal expression of atumor-associated antigen identified according to the invention. Inparticular embodiments, said agent may cause induction of cell death,reduction in cell growth, damage to the cell membrane or secretion ofcytokines and preferably have a tumor-inhibiting activity. In oneembodiment, the agent is an antisense nucleic acid which hybridizesselectively with the nucleic acid coding for the tumor-associatedantigen. In a further embodiment, the agent is an antibody which bindsselectively to the tumor-associated antigen, in particular acomplement-activated or toxin conjugated antibody which bindsselectively to the tumor-associated antigen. In a further embodiment,the agent comprises two or more agents which each selectively recognizedifferent tumor-associated antigens, at least one of which is atumor-associated antigen identified according to the invention.Recognition needs not be accompanied directly with inhibition ofactivity or expression of the antigen. In this aspect of the invention,the antigen selectively limited to tumors preferably serves as a labelfor recruiting effector mechanisms to this specific location. In apreferred embodiment, the agent is a cytotoxic T lymphocyte whichrecognizes the antigen on an HLA molecule and lyses the cells labeled inthis way. In a further embodiment, the agent is an antibody which bindsselectively to the tumor-associated antigen and thus recruits natural orartificial effector mechanisms to said cell. In a further embodiment,the agent is a T helper lymphocyte which enhances effector functions ofother cells specifically recognizing said antigen.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which inhibits expression or activity of atumor-associated antigen identified according to the invention. In apreferred embodiment, the agent is an antisense nucleic acid whichhybridizes selectively with the nucleic acid coding for thetumor-associated antigen. In a further embodiment, the agent is anantibody which binds selectively to the tumor-associated antigen. In afurther embodiment, the agent comprises two or more agents which eachselectively inhibit expression or activity of different tumor-associatedantigens, at least one of which is a tumor-associated antigen identifiedaccording to the invention.

The invention furthermore relates to a pharmaceutical composition whichcomprises an agent which, when administered, selectively increases theamount of complexes between an HLA molecule and a peptide epitope fromthe tumor-associated antigen identified according to the invention. Inone embodiment, the agent comprises one or more components selected fromthe group consisting of (i) the tumor-associated antigen or a partthereof, (ii) a nucleic acid which codes for said tumor-associatedantigen or a part thereof, (iii) a host cell which expresses saidtumor-associated antigen or a part thereof, and (iv) isolated complexesbetween peptide epitopes from said tumor-associated antigen and an MHCmolecule. In one embodiment, the agent comprises two or more agentswhich each selectively increase the amount of complexes between MHCmolecules and peptide epitopes of different tumor-associated antigens,at least one of which is a tumor-associated antigen identified accordingto the invention.

The invention furthermore relates to a pharmaceutical composition whichcomprises one or more components selected from the group consisting of(i) a tumor-associated antigen identified according to the invention ora part thereof, (ii) a nucleic acid which codes for a tumor-associatedantigen identified according to the invention or for a part thereof,(iii) an antibody which binds to a tumor-associated antigen identifiedaccording to the invention or to a part thereof, (iv) an antisensenucleic acid which hybridizes specifically with a nucleic acid codingfor a tumor-associated antigen identified according to the invention,(v) a host cell which expresses a tumor-associated antigen identifiedaccording to the invention or a part thereof, and (vi) isolatedcomplexes between a tumor-associated antigen identified according to theinvention or a part thereof and an HLA molecule.

A nucleic acid coding for a tumor-associated antigen identifiedaccording to the invention or for a part thereof may be present in thepharmaceutical composition in an expression vector and functionallylinked to a promoter.

A host cell present in a pharmaceutical composition of the invention maysecrete the tumor-associated antigen or the part thereof, express it onthe surface or may additionally express an HLA molecule which binds tosaid tumor-associated antigen or said part thereof. In one embodiment,the host cell expresses the HLA molecule endogenously. In a furtherembodiment, the host cell expresses the HLA molecule and/or thetumor-associated antigen or the part thereof in a recombinant manner.The host cell is preferably nonproliferative. In a preferred embodiment,the host cell is an antigen-presenting cell, in particular a dendriticcell, a monocyte or a macrophage.

An antibody present in a pharmaceutical composition of the invention maybe a monoclonal antibody. In further embodiments, the antibody is achimeric or humanized antibody, a fragment of a natural antibody or asynthetic antibody, all of which may be produced by combinatorytechniques. The antibody may be coupled to a therapeutically ordiagnostically useful agent.

An antisense nucleic acid present in a pharmaceutical composition of theinvention may comprise a sequence of 6-50, in particular 10-30, 15-30and 20-30, contiguous nucleotides of the nucleic acid coding for thetumor-associated antigen identified according to the invention.

In further embodiments, a tumor-associated antigen, provided by apharmaceutical composition of the invention either directly or viaexpression of a nucleic acid, or a part thereof binds to MHC moleculeson the surface of cells, said binding preferably causing a cytolyticresponse and/or inducing cytokine release.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier and/or an adjuvant. The adjuvant maybe selected from saponin, GM-CSF, CpG nucleotides, RNA, a cytokine or achemokine. A pharmaceutical composition of the invention is preferablyused for the treatment of a disease characterized by selectiveexpression or abnormal expression of a tumor-associated antigen. In apreferred embodiment, the disease is cancer.

The invention furthermore relates to methods of treating or diagnosing adisease characterized by expression or abnormal expression of one ofmore tumor-associated antigens. In one embodiment, the treatmentcomprises administering a pharmaceutical composition of the invention.

In one aspect, the invention relates to a method of diagnosing a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention. The method comprisesdetection of (i) a nucleic acid which codes for the tumor-associatedantigen or of a part thereof and/or (ii) detection of thetumor-associated antigen or of a part thereof, and/or (iii) detection ofan antibody to the tumor-associated antigen or to a part thereof and/or(iv) detection of cytotoxic or T helper lymphocytes which are specificfor the tumor-associated antigen or for a part thereof in a biologicalsample isolated from a patient. In particular embodiments, detectioncomprises (i) contacting the biological sample with an agent which bindsspecifically to the nucleic acid coding for the tumor-associated antigenor to the part thereof, to said tumor-associated antigen or said partthereof, to the antibody or to cytotoxic or T helper lymphocytesspecific for the tumor-associated antigen or parts thereof, and (ii)detecting the formation of a complex between the agent and the nucleicacid or the part thereof, the tumor-associated antigen or the partthereof, the antibody or the cytotoxic or T helper lymphocytes. In oneembodiment, the disease is characterized by expression or abnormalexpression of two or more different tumor-associated antigens anddetection comprises detection of two or more nucleic acids coding forsaid two or more different tumor-associated antigens or of partsthereof, detection of two or more different tumor-associated antigens orof parts thereof, detection of two or more antibodies binding to saidtwo or more different tumor-associated antigens or to parts thereof ordetection of two or more cytotoxic or T helper lymphocytes specific forsaid two or more different tumor-associated antigens. In a furtherembodiment, the biological sample isolated from the patient is comparedto a comparable normal biological sample.

In a further aspect, the invention relates to a method for determiningregression, course or onset of a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the invention, which method comprises monitoring a sample from apatient who has said disease or is suspected of falling ill with saiddisease, with respect to one or more parameters selected from the groupconsisting of (i) the amount of nucleic acid which codes for thetumor-associated antigen or of a part thereof, (ii) the amount of thetumor-associated antigen or a part thereof, (iii) the amount ofantibodies which bind to the tumor-associated antigen or to a partthereof, and (iv) the amount of cytolytic T cells or T helper cellswhich are specific for a complex between the tumor-associated antigen ora part thereof and an MHC molecule. The method preferably comprisesdetermining the parameter(s) in a first sample at a first point in timeand in a further sample at a second point in time and in which thecourse of the disease is determined by comparing the two samples. Inparticular embodiments, the disease is characterized by expression orabnormal expression of two or more different tumor-associated antigensand monitoring comprises monitoring (i) the amount of two or morenucleic acids which code for said two or more different tumor-associatedantigens or of parts thereof, and/or (ii) the amount of said two or moredifferent tumor-associated antigens or of parts thereof, and/or (iii)the amount of two or more antibodies which bind to said two or moredifferent tumor-associated antigens or to parts thereof, and/or (iv) theamount of two or more cytolytic T cells or of T helper cells which arespecific for complexes between said two or more differenttumor-associated antigens or of parts thereof and MHC molecules.

According to the invention, detection of a nucleic acid or of a partthereof or monitoring the amount of a nucleic acid or of a part thereofmay be carried out using a polynucleotide probe which hybridizesspecifically to said nucleic acid or said part thereof or may be carriedout by selective amplification of said nucleic acid or said partthereof. In one embodiment, the polynucleotide probe comprises asequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguousnucleotides of said nucleic acid.

In particular embodiments, the tumor-associated antigen to be detectedor the part thereof is present intracellularly or on the cell surface.According to the invention, detection of a tumor-associated antigen orof a part thereof or monitoring the amount of a tumor-associated antigenor of a part thereof may be carried out using an antibody bindingspecifically to said tumor-associated antigen or said part thereof.

In further embodiments, the tumor-associated antigen to be detected orthe part thereof is present in a complex with an MHC molecule, inparticular an HLA molecule.

According to the invention, detection of an antibody or monitoring theamount of antibodies may be carried out using a protein or peptidebinding specifically to said antibody.

According to the invention, detection of cytolytic T cells or of Thelper cells or monitoring the amount of cytolytic T cells or of Thelper cells which are specific for complexes between an antigen or apart thereof and MHC molecules may be carried out using a cellpresenting the complex between said antigen or said part thereof and anMHC molecule.

The polynucleotide probe, the antibody, the protein or peptide or thecell, which is used for detection or monitoring, is preferably labeledin a detectable manner. In particular embodiments, the detectable markeris a radioactive marker or an enzymic marker. T lymphocytes mayadditionally be detected by detecting their proliferation, theircytokine production, and their cytotoxic activity triggered by specificstimulation with the complex of MHC and tumor-associated antigen orparts thereof. T lymphocytes may also be detected via a recombinant MHCmolecule or else a complex of two or more MHC molecules which are loadedwith the particular immunogenic fragment of one or more of thetumor-associated antigens and which can identify the specific Tlymphocytes by contacting the specific T cell receptor.

In a further aspect, the invention relates to a method of treating,diagnosing or monitoring a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the invention, which method comprises administering an antibody whichbinds to said tumor-associated antigen or to a part thereof and which iscoupled to a therapeutic or diagnostic agent. The antibody may be amonoclonal antibody. In further embodiments, the antibody is a chimericor humanized antibody or a fragment of a natural antibody.

The invention also relates to a method of treating a patient having adisease characterized by expression or abnormal expression of atumor-associated antigen identified according to the invention, whichmethod comprises (i) removing a sample containing immunoreactive cellsfrom said patient, (ii) contacting said sample with a host cellexpressing said tumor-associated antigen or a part thereof, underconditions which favor production of cytolytic T cells against saidtumor-associated antigen or a part thereof, and (iii) introducing thecytolytic T cells into the patient in an amount suitable for lysingcells expressing the tumor-associated antigen or a part thereof. Theinvention likewise relates to cloning the T cell receptor of cytolytic Tcells against the tumor-associated antigen. Said receptor may betransferred to other T cells which thus receive the desired specificityand, as under (iii), may be introduced into the patient.

In one embodiment, the host cell endogenously expresses an HLA molecule.In a further embodiment, the host cell recombinantly expresses an HLAmolecule and/or the tumor-associated antigen or the part thereof. Thehost cell is preferably nonproliferative. In a preferred embodiment, thehost cell is an antigen-presenting cell, in particular a dendritic cell,a monocyte or a macrophage.

In a further aspect, the invention relates to a method of treating apatient having a disease characterized by expression or abnormalexpression of a tumor-associated antigen, which method comprises (i)identifying a nucleic acid which codes for a tumor-associated antigenidentified according to the invention and which is expressed by cellsassociated with said disease, (ii) transfecting a host cell with saidnucleic acid or a part thereof, (iii) culturing the transfected hostcell for expression of said nucleic acid (this is not obligatory when ahigh rate of transfection is obtained), and (iv) introducing the hostcells or an extract thereof into the patient in an amount suitable forincreasing the immune response to the patient's cells associated withthe disease. The method may further comprise identifying an MHC moleculepresenting the tumor-associated antigen or a part thereof, with the hostcell expressing the identified MHC molecule and presenting saidtumor-associated antigen or a part thereof. The immune response maycomprise a B cell response or a T cell response. Furthermore, a T cellresponse may comprise production of cytolytic T cells and/or T helpercells which are specific for the host cells presenting thetumor-associated antigen or a part thereof or specific for cells of thepatient which express said tumor-associated antigen or a part thereof.

The invention also relates to a method of treating a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention, which method comprises(i) identifying cells from the patient which express abnormal amounts ofthe tumor-associated antigen, (ii) isolating a sample of said cells,(iii) culturing said cells, and (iv) introducing said cells into thepatient in an amount suitable for triggering an immune response to thecells.

Preferably, the host cells used according to the invention arenonproliferative or are rendered nonproliferative. A diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen is in particular cancer.

The present invention furthermore relates to a nucleic acid selectedfrom the group consisting of (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:3-5, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c). The invention furthermore relates to a nucleic acid,which codes for a protein or polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 10 and 12-14,a part or derivative thereof.

In a further aspect, the invention relates to promoter sequences ofnucleic acids of the invention. These sequences may be functionallylinked to another gene, preferably in an expression vector, and thusensure selective expression of said gene in appropriate cells.

In a further aspect, the invention relates to a recombinant nucleic acidmolecule, in particular DNA or RNA molecule, which comprises a nucleicacid of the invention.

The invention also relates to host cells which contain a nucleic acid ofthe invention or a recombinant nucleic acid molecule comprising anucleic acid of the invention.

The host cell may also comprise a nucleic acid coding for a HLAmolecule. In one embodiment, the host cell endogenously expresses theHLA molecule. In a further embodiment, the host cell recombinantlyexpresses the HLA molecule and/or the nucleic acid of the invention or apart thereof. Preferably, the host cell is nonproliferative. In apreferred embodiment, the host cell is an antigen-presenting cell, inparticular a dendritic cell, a monocyte or a macrophage.

In a further embodiment, the invention relates to oligonucleotides whichhybridize with a nucleic acid identified according to the invention andwhich may be used as genetic probes or as “antisense” molecules. Nucleicacid molecules in the form of oligonucleotide primers or competentsamples, which hybridize with a nucleic acid identified according to theinvention or parts thereof, may be used for finding nucleic acids whichare homologous to said nucleic acid identified according to theinvention. PCR amplification, Southern and Northern hybridization may beemployed for finding homologous nucleic acids. Hybridization may becarried out under low stringency, more preferably under mediumstringency and most preferably under high stringency conditions. Theterm “stringent conditions” according to the invention refers toconditions which allow specific hybridization between polynucleotides.

In a further aspect, the invention relates to a protein, polypeptide orpeptide which is encoded by a nucleic acid selected from the groupconsisting of (a) a nucleic acid which comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 3-5, a part orderivative thereof, (b) a nucleic acid which hybridizes with the nucleicacid of (a) under stringent conditions, (c) a nucleic acid which isdegenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c). In a preferred embodiment, the invention relates to a protein orpolypeptide or peptide which comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 10 and 12-14, a part orderivative thereof.

In a further aspect, the invention relates to an immunogenic fragment ofa tumor-associated antigen identified according to the invention. Saidfragment preferably binds to a human HLA receptor or to a humanantibody. A fragment of the invention preferably comprises a sequence ofat least 6, in particular at least 8, at least 10, at least 12, at least15, at least 20, at least 30 or at least 50, amino acids.

In this aspect the invention relates, in particular, to a peptide whichhas or comprises a sequence selected from the group consisting of SEQ IDNOs: 17-19, 90-97, 100-102, 105, 106, 111-116, 120, 123, 124, and135-137, a part or derivative thereof.

In a further aspect, the invention relates to an agent which binds to atumor-associated antigen identified according to the invention or to apart thereof. In a preferred embodiment, the agent is an antibody. Infurther embodiments, the antibody is a chimeric, a humanized antibody oran antibody produced by combinatory techniques or is a fragment of anantibody. Furthermore, the invention relates to an antibody which bindsselectively to a complex of (i) a tumor-associated antigen identifiedaccording to the invention or a part thereof and (ii) an MHC molecule towhich said tumor-associated antigen identified according to theinvention or said part thereof binds, with said antibody not binding to(i) or (ii) alone. An antibody of the invention may be a monoclonalantibody. In further embodiments, the antibody is a chimeric orhumanized antibody or a fragment of a natural antibody.

In particular, the invention relates to such an agent, in particular anantibody, which specifically binds to a peptide which has or comprises asequence selected from the group consisting of SEQ ID NOs: 17-19, 90-97,100-102, 105, 106, 111-116, 120, 123, 124, and 135-137, a part orderivative thereof.

The invention furthermore relates to a conjugate between an agent of theinvention which binds to a tumor-associated antigen identified accordingto the invention or to a part thereof or an antibody of the inventionand a therapeutic or diagnostic agent. In one embodiment, thetherapeutic or diagnostic agent is a toxin.

In a further aspect, the invention relates to a kit for detectingexpression or abnormal expression of a tumor-associated antigenidentified according to the invention, which kit comprises agents fordetection (i) of the nucleic acid which codes for the tumor-associatedantigen or of a part thereof, (ii) of the tumor-associated antigen or ofa part thereof, (iii) of antibodies which bind to the tumor-associatedantigen or to a part thereof, and/or (iv) of T cells which are specificfor a complex between the tumor-associated antigen or a part thereof andan MHC molecule. In one embodiment, the agents for detection of thenucleic acid or the part thereof are nucleic acid molecules forselective amplification of said nucleic acid, which comprise, inparticular a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of said nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, genes are described which are expressed intumor cells selectively or aberrantly and which are tumor-associatedantigens.

According to the invention, these genes and/or their genetic productsand/or their derivatives and/or parts are preferred target structuresfor therapeutic approaches. Conceptionally, said therapeutic approachesmay aim at inhibiting the activity of the selectively expressedtumor-associated genetic product. This is useful, if said aberrantrespective selective expression is functionally important in tumorpathogenecity and if its ligation is accompanied by selective damage ofthe corresponding cells. Other therapeutic concepts contemplatetumor-associated antigens as labels which recruit effector mechanismshaving cell-damaging potential selectively to tumor cells. Here, thefunction of the target molecule itself and its role in tumor developmentare totally irrelevant.

“Derivative” of a nucleic acid means according to the invention thatsingle or multiple nucleotide substitutions, deletions and/or additionsare present in said nucleic acid. Furthermore, the term “derivative”also comprises chemical derivatization of a nucleic acid on a nucleotidebase, on the sugar or on the phosphate. The term “derivative” alsocomprises nucleic acids which contain nucleotides and nucleotide analogsnot occurring naturally.

According to the invention, a nucleic acid is preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidscomprise according to the invention genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the invention, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences are capable of hybridizing and forming a stable duplex withone another, with hybridization preferably being carried out underconditions which allow specific hybridization between polynucleotides(stringent conditions). Stringent conditions are described, for example,in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel etal., Editors, John Wiley & Sons, Inc., New York and refer, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15M sodium citrate, pH 7. After hybridization, the membrane to which theDNA has been transferred is washed, for example, in 2×SSC at roomtemperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68°C.

According to the invention, complementary nucleic acids have at least40%, in particular at least 50%, at least 60%, at least 70%, at least80%, at least 90% and preferably at least 95%, at least 98% or at least99%, identical nucleotides.

Nucleic acids coding for tumor-associated antigens may, according to theinvention, be present alone or in combination with other nucleic acids,in particular heterologous nucleic acids. In preferred embodiments, anucleic acid is functionally linked to expression control sequences orregulatory sequences which may be homologous or heterologous withrespect to said nucleic acid. A coding sequence and a regulatorysequence are “functionally” linked to one another, if they arecovalently linked to one another in such a way that expression ortranscription of said coding sequence is under the control or under theinfluence of said regulatory sequence. If the coding sequence is to betranslated into a functional protein, then, with a regulatory sequencefunctionally linked to said coding sequence, induction of saidregulatory sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or said codingsequence not being capable of being translated into the desired proteinor peptide.

The term “expression control sequence” or “regulatory sequence”comprises according to the invention promoters, enhancers and othercontrol elements which regulate expression of a gene. In particularembodiments of the invention, the expression control sequences can beregulated. The exact structure of regulatory sequences may vary as afunction of the species or cell type, but generally comprises5′untranscribed and 5′untranslated sequences which are involved ininitiation of transcription and translation, respectively, such as TATAbox, capping sequence, CAAT sequence, and the like. More specifically,5′untranscribed regulatory sequences comprise a promoter region whichincludes a promoter sequence for transcriptional control of thefunctionally linked gene. Regulatory sequences may also compriseenhancer sequences or upstream activator sequences.

Thus, on the one hand, the tumor-associated antigens illustrated hereinmay be combined with any expression control sequences and promoters. Onthe other hand, however, the promoters of the tumor-associated geneticproducts illustrated herein may, according to the invention, be combinedwith any other genes. This allows the selective activity of thesepromoters to be utilized.

According to the invention, a nucleic acid may furthermore be present incombination with another nucleic acid which codes for a polypeptidecontrolling secretion of the protein or polypeptide encoded by saidnucleic acid from a host cell. According to the invention, a nucleicacid may also be present in combination with another nucleic acid whichcodes for a polypeptide causing the encoded protein or polypeptide to beanchored on the cell membrane of the host cell or compartmentalized intoparticular organelles of said cell. Similarly, a combination with anucleic acid is possible which represents a reporter gene or any “tag”.

In a preferred embodiment, a recombinant DNA molecule is according tothe invention a vector, where appropriate with a promoter, whichcontrols expression of a nucleic acid, for example a nucleic acid codingfor a tumor-associated antigen of the invention. The term “vector” isused here in its most general meaning and comprises any intermediaryvehicle for a nucleic acid which enables said nucleic acid, for example,to be introduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. An intermediaryvehicle may be adapted, for example, to the use in electroporation, inbombardment with microprojectiles, in liposomal administration, in thetransfer with the aid of agrobacteria or in insertion via DNA or RNAviruses. Vectors comprise plasmids, phagemids or viral genomes.

The nucleic acids coding for a tumor-associated antigen identifiedaccording to the invention may be used for transfection of host cells.Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNAmay be prepared by in-vitro transcription of a DNA template.Furthermore, it may be modified by stabilizing sequences, capping andpolyadenylation prior to application.

According to the invention, the term “host cell” relates to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cells” comprises according to the invention prokaryotic(e.g. E. coli) or eukaryotic cells (e.g. dendritic cells, B cells, CHOcells, COS cells, K562 cells, yeast cells and insect cells). Particularpreference is given to mammalian cells such as cells from humans, mice,hamsters, pigs, goats, primates. The cells may be derived from amultiplicity of tissue types and comprise primary cells and cell lines.

Specific examples comprise keratinocytes, peripheral blood leukocytes,stem cells of the bone marrow and embryonic stem cells. In furtherembodiments, the host cell is an antigen-presenting cell, in particulara dendritic cell, monocyte or a macrophage. A nucleic acid may bepresent in the host cell in the form of a single copy or of two or morecopies and, in one embodiment, is expressed in the host cell.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.Preferred expression systems in mammalian cells comprise pcDNA3.1 andpRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selectablemarker such as a gene imparting resistance to G418 (and thus enablingstably transfected cell lines to be selected) and the enhancer-promotersequences of cytomegalovirus (CMV).

In those cases of the invention in which an HLA molecule presents atumor-associated antigen or a part thereof, an expression vector mayalso comprise a nucleic acid sequence coding for said HLA molecule. Thenucleic acid sequence coding for the HLA molecule may be present on thesame expression vector as the nucleic acid coding for thetumor-associated antigen or the part thereof, or both nucleic acids maybe present on different expression vectors. In the latter case, the twoexpression vectors may be cotransfected into a cell. If a host cellexpresses neither the tumor-associated antigen or the part thereof northe HLA molecule, both nucleic acids coding therefor are transfectedinto the cell either on the same expression vector or on differentexpression vectors. If the cell already expresses the HLA molecule, onlythe nucleic acid sequence coding for the tumor-associated antigen or thepart thereof can be transfected into the cell.

The invention also comprises kits for amplification of a nucleic acidcoding for a tumor-associated antigen. Such kits comprise, for example,a pair of amplification primers which hybridize to the nucleic acidcoding for the tumor-associated antigen. The primers preferably comprisea sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguousnucleotides of the nucleic acid and are nonoverlapping, in order toavoid the formation of primer dimers. One of the primers will hybridizeto one strand of the nucleic acid coding for the tumor-associatedantigen, and the other primer will hybridize to the complementary strandin an arrangement which allows amplification of the nucleic acid codingfor the tumor-associated antigen.

“Antisense” molecules or “antisense” nucleic acids may be used forregulating, in particular reducing, expression of a nucleic acid. Theterm “antisense molecule” or “antisense nucleic acid” refers accordingto the invention to an oligonucleotide which is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide or modifiedoligodeoxyribonucleotide and which hybridizes under physiologicalconditions to DNA comprising a particular gene or to mRNA of said gene,thereby inhibiting transcription of said gene and/or translation of saidmRNA. According to the invention, an “antisense molecule” also comprisesa construct which contains a nucleic acid or a part thereof in reverseorientation with respect to its natural promoter. An antisensetranscript of a nucleic acid or of a part thereof may form a duplex withthe naturally occurring mRNA specifying the enzyme and thus preventaccumulation of or translation of the mRNA into the active enzyme.

Another possibility is the use of ribozymes for inactivating a nucleicacid. Antisense oligonucleotides preferred according to the inventionhave a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of the target nucleic acid and preferably arefully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes withan N-terminal or 5′ upstream site such as a translation initiation site,transcription initiation site or promoter site. In further embodiments,the antisense oligonucleotide hybridizes with a 3′untranslated region ormRNA splicing site.

In one embodiment, an oligonucleotide of the invention consists ofribonucleotides, deoxyribonucleotides or a combination thereof, with the5′ end of one nucleotide and the 3′ end of another nucleotide beinglinked to one another by a phosphodiester bond. These oligonucleotidesmay be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the invention is a“modified” oligonucleotide. Here, the oligonucleotide may be modified invery different ways, without impairing its ability to bind its target,in order to increase, for example, its stability or therapeuticefficacy. According to the invention, the term “modifiedoligonucleotide” means an oligonucleotide in which (i) at least two ofits nucleotides are linked to one another by a synthetic internucleosidebond (i.e. an internucleoside bond which is not a phosphodiester bond)and/or (ii) a chemical group which is usually not found in nucleic acidsis covalently linked to the oligonucleotide. Preferred syntheticinternucleoside bonds are phosphorothioates, alkyl phosphonates,phosphorodithioates, phosphate esters, alkyl phosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotideshaving a covalently modified base and/or sugar. “Modifiedoligonucleotides” comprise, for example, oligonucleotides with sugarresidues which are covalently bound to low molecular weight organicgroups other than a hydroxyl group at the 3′ position and a phosphategroup at the 5′ position. Modified oligonucleotides may comprise, forexample, a 2′-O-alkylated ribose residue or another sugar instead ofribose, such as arabinose.

Preferably, the proteins and polypeptides described according to theinvention have been isolated. The terms “isolated protein” or “isolatedpolypeptide” mean that the protein or polypeptide has been separatedfrom its natural environment. An isolated protein or polypeptide may bein an essentially purified state. The term “essentially purified” meansthat the protein or polypeptide is essentially free of other substanceswith which it is associated in nature or in vivo.

Such proteins and polypeptides may be used, for example, in producingantibodies and in an immunological or diagnostic assay or astherapeutics. Proteins and polypeptides described according to theinvention may be isolated from biological samples such as tissue or cellhomogenates and may also be expressed recombinantly in a multiplicity ofpro- or eukaryotic expression systems.

For the purposes of the present invention, “derivatives” of a protein orpolypeptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible. Amino acid deletion variants are characterized by theremoval of one or more amino acids from the sequence. Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpolypeptides. Preference is given to replacing amino acids with otherones having similar properties such as hydrophobicity, hydrophilicity,electronegativity, volume of the side chain and the like (conservativesubstitution). Conservative substitutions, for example, relate to theexchange of one amino acid with another amino acid listed below in thesame group as the amino acid to be substituted:

-   -   1. small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly)    -   2. negatively charged residues and their amides: Asn, Asp, Glu,        Gln    -   3. positively charged residues: His, Arg, Lys    -   4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)    -   5. large aromatic residues: Phe, Tyr, Trp.

Owing to their particular part in protein architecture, three residuesare shown in brackets. Gly is the only residue without a side chain andthus imparts flexibility to the chain. Pro has an unusual geometry whichgreatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. Techniques for introducing substitution mutations atpredetermined sites into DNA which has a known or partially knownsequence are well known and comprise M13 mutagenesis, for example. Themanipulation of DNA sequences for preparing proteins havingsubstitutions, insertions or deletions, is described in detail inSambrook et al. (1989), for example.

According to the invention, “derivatives” of proteins, polypeptides orpeptides also comprise single or multiple substitutions, deletionsand/or additions of any molecules associated with the enzyme, such ascarbohydrates, lipids and/or proteins, polypeptides or peptides. Theterm “derivative” also extends to all functional chemical equivalents ofsaid proteins, polypeptides or peptides.

According to the invention, a part or fragment of a tumor-associatedantigen has a functional property of the polypeptide from which it hasbeen derived. Such functional properties comprise the interaction withantibodies, the interaction with other polypeptides or proteins, theselective binding of nucleic acids and an enzymatic activity. Aparticular property is the ability to form a complex with HLA and, whereappropriate, generate an immune response. This immune response may bebased on stimulating cytotoxic or T helper cells. A part or fragment ofa tumor-associated antigen of the invention preferably comprises asequence of at least 6, in particular at least 8, at least 10, at least12, at least 15, at least 20, at least 30 or at least 50, consecutiveamino acids of the tumor-associated antigen.

A part or a fragment of a nucleic acid coding for a tumor-associatedantigen relates according to the invention to the part of the nucleicacid, which codes at least for the tumor-associated antigen and/or for apart or a fragment of said tumor-associated antigen, as defined above.

The isolation and identification of genes coding for tumor-associatedantigens also make possible the diagnosis of a disease characterized byexpression of one or more tumor-associated antigens. These methodscomprise determining one or more nucleic acids which code for atumor-associated antigen and/or determining the encoded tumor-associatedantigens and/or peptides derived therefrom. The nucleic acids may bedetermined in the conventional manner, including by polymerase chainreaction or hybridization with a labeled probe. Tumor-associatedantigens or peptides derived therefrom may be determined by screeningpatient antisera with respect to recognizing the antigen and/or thepeptides. They may also be determined by screening T cells of thepatient for specificities for the corresponding tumor-associatedantigen.

The present invention also enables proteins binding to tumor-associatedantigens described herein to be isolated, including antibodies andcellular binding partners of said tumor-associated antigens.

According to the invention, particular embodiments ought to involveproviding “dominant negative” polypeptides derived from tumor-associatedantigens. A dominant negative polypeptide is an inactive protein variantwhich, by way of interacting with the cellular machinery, displaces anactive protein from its interaction with the cellular machinery or whichcompetes with the active protein, thereby reducing the effect of saidactive protein. For example, a dominant negative receptor which binds toa ligand but does not generate any signal as response to binding to theligand can reduce the biological effect of said ligand. Similarly, adominant negative catalytically inactive kinase which usually interactswith target proteins but does not phosphorylate said target proteins mayreduce phosphorylation of said target proteins as response to a cellularsignal. Similarly, a dominant negative transcription factor which bindsto a promoter site in the control region of a gene but does not increasetranscription of said gene may reduce the effect of a normaltranscription factor by occupying promoter binding sites, withoutincreasing transcription.

The result of expression of a dominant negative polypeptide in a cell isa reduction in the function of active proteins. The skilled worker mayprepare dominant negative variants of a protein, for example, byconventional mutagenesis methods and by evaluating the dominant negativeeffect of the variant polypeptide.

The invention also comprises substances such as polypeptides which bindto tumor-associated antigens. Such binding substances may be used, forexample, in screening assays for detecting tumor-associated antigens andcomplexes of tumor-associated antigens with their binding partners andin the purification of said tumor-associated antigens and of complexesthereof with their binding partners. Such substances may also be usedfor inhibiting the activity of tumor-associated antigens, for example bybinding to such antigens.

The invention therefore comprises binding substances such as, forexample, antibodies or antibody fragments, which are capable ofselectively binding to tumor-associated antigens. Antibodies comprisepolyclonal and monoclonal antibodies which are produced in theconventional manner.

Such antibodies can recognize proteins in the native and/or denaturatedstate (Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J.Immunol. Methods 234: 107-116, 2000; Kayyem et al., Eur. J. Biochem.208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224: 51-60, 1999).

Antisera which contain specific antibodies specifically binding to thetarget protein can be prepared by various standard processes; see, forexample, “Monoclonal Antibodies: A Practical Approach” by PhilipShepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A LaboratoryManual” by Ed Harlow, David Lane, ISBN: 0879693142 and “UsingAntibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow,David Lane, Ed Harlow ISBN 0879695447. Thereby it is also possible togenerate affine and specific antibodies which recognize complex membraneproteins in their native form (Azorsa et al., J. Immunol. Methods 229:35-48, 1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989;Gardsvoll, J. Immunol. Methods 234: 107-116, 2000). This is inparticular relevant for the preparation of antibodies which are to beused therapeutically, but also for many diagnostic applications. In thisrespect, it is possible to immunize with the whole protein, withextracellular partial sequences as well as with cells which express thetarget molecule in physiologically folded form.

Monoclonal antibodies are traditionally prepared using the hybridomatechnology. (for technical details see: “Monoclonal Antibodies: APractical Approach” by Philip Shepherd, Christopher Dean ISBN0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, DavidLane ISBN: 0879693142; “Using Antibodies: A Laboratory Manual: PortableProtocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).

It is known that only a small part of an antibody molecule, theparatope, is involved in binding of the antibody to its epitope (cf.Clark, W. R. (1986), The Experimental Foundations of Modern Immunology,Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology,7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fcregions are, for example, effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pfc′ regionhas been enzymatically removed or which has been produced without thepFc′ region, referred to as F(ab′)₂ fragment, carries both antigenbinding sites of a complete antibody. Similarly, an antibody from whichthe Fc region has been enzymatically removed or which has been producedwithout said Fc region, referred to as Fab fragment, carries one antigenbinding site of an intact antibody molecule. Furthermore, Fab fragmentsconsist of a covalently bound light chain of an antibody and part of theheavy chain of said antibody, referred to as Fd. The Fd fragments arethe main determinants of antibody specificity (a single Fd fragment canbe associated with up to ten different light chains, without alteringthe specificity of the antibody) and Fd fragments, when isolated, retainthe ability to bind to an epitope.

Located within the antigen-binding part of an antibody arecomplementary-determining regions (CDRs) which interact directly withthe antigen epitope and framework regions (FRs) which maintain thetertiary structure of the paratope. Both the Fd fragment of the heavychain and the light chain of IgG immunoglobulins contain four frameworkregions (FR1 to FR4) which are separated in each case by threecomplementary-determining regions (CDR1 to CDR3). The CDRs and, inparticular, the CDR3 regions and, still more particularly, the CDR3region of the heavy chain are responsible to a large extent for antibodyspecificity.

Non-CDR regions of a mammalian antibody are known to be able to bereplaced by similar regions of antibodies with the same or a differentspecificity, with the specificity for the epitope of the originalantibody being retained. This made possible the development of“humanized” antibodies in which nonhuman CDRs are covalently linked tohuman FR and/or Fc/pFc′ regions to produce a functional antibody.

This is utilized in the so called “SLAM” technology, wherein B cellsfrom whole blood are isolated and the cells are monocloned. Then, thesupernatant of the single B cells is analyzed with respect to itsantibody specificity. In contrast to the hybridoma technology thevariable region of the antibody gene is amplified using single cell PCRand cloned into a suitable vector. In this way, the provision ofmonoclonal antibodies is accelerated (de Wildt et al., J. Immunol.Methods 207: 61-67, 1997).

As another example, WO 92/04381 describes the production and use ofhumanized murine RSV antibodies in which at least part of the murine FRregions have been replaced with FR regions of a human origin. Antibodiesof this kind, including fragments of intact antibodies withantigen-binding capability, are often referred to as “chimeric”antibodies.

The invention also provides F(ab′)₂, Fab, Fv, and Fd fragments ofantibodies, chimeric antibodies, in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, chimeric F(ab′)₂-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain-CDR3 regions have been replaced with homologous human or nonhumansequences, chimeric Fab-fragment antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, and chimeric Fd-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 regions have beenreplaced with homologous human or nonhuman sequences. The invention alsocomprises “single-chain” antibodies.

The invention also comprises polypeptides which bind specifically totumor-associated antigens. Polypeptide binding substances of this kindmay be provided, for example, by degenerate peptide libraries which maybe prepared simply in solution in an immobilized form or asphage-display libraries. It is likewise possible to preparecombinatorial libraries of peptides with one or more amino acids.Libraries of peptoids and nonpeptidic synthetic residues may also beprepared.

Phage display may be particularly effective in identifying bindingpeptides of the invention. In this connection, for example, a phagelibrary is prepared (using, for example, the M13, fd or lambda phages)which presents inserts of from 4 to about 80 amino acid residues inlength. Phages are then selected which carry inserts which bind to thetumor-associated antigen. This process may be repeated via two or morecycles of a reselection of phages binding to the tumor-associatedantigen. Repeated rounds result in a concentration of phages carryingparticular sequences. An analysis of DNA sequences may be carried out inorder to identify the sequences of the expressed polypeptides. Thesmallest linear portion of the sequence binding to the tumor-associatedantigen may be determined. The “two-hybrid system” of yeast may also beused for identifying polypeptides which bind to a tumor-associatedantigen. Tumor-associated antigens described according to the inventionor fragments thereof may be used for screening peptide libraries,including phage-display libraries, in order to identify and selectpeptide binding partners of the tumor-associated antigens. Suchmolecules may be used, for example, for screening assays, purificationprotocols, for interference with the function of the tumor-associatedantigen and for other purposes known to the skilled worker.

The antibodies described above and other binding molecules may be used,for example, for identifying tissue which expresses a tumor-associatedantigen. Antibodies may also be coupled to specific diagnosticsubstances for displaying cells and tissues expressing tumor-associatedantigens. They may also be coupled to therapeutically useful substances.Diagnostic substances comprise, in a nonlimiting manner, barium sulfate,iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate,meglumine diatrizoate, metrizamide, sodium tyropanoate and radiodiagnostic, including positron emitters such as fluorine-18 andcarbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131and indium-111, nuclides for nuclear magnetic resonance, such asfluorine and gadolinium. According to the invention, the term“therapeutically useful substance” means any therapeutic molecule which,as desired, is selectively guided to a cell which expresses one or moretumor-associated antigens, including anticancer agents, radioactiveiodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc.Anticancer agents comprise, for example, aminoglutethimide,azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine,dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil,interferon-α, lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HCl, thioguanine, vinblastine sulfate and vincristinesulfate. Other anticancer agents are described, for example, in Goodmanand Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition,1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such aspokeweed antiviral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxinresidues may also be high energy-emitting radionuclides such ascobalt-60.

The term “patient” means according to the invention a human being, anonhuman primate or another animal, in particular a mammal such as acow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse andrat. In a particularly preferred embodiment, the patient is a humanbeing.

According to the invention, the term “disease” refers to anypathological state in which tumor-associated antigens are expressed orabnormally expressed. “Abnormal expression” means according to theinvention that expression is altered, preferably increased, compared tothe state in a healthy individual. An increase in expression refers toan increase by at least 10%, in particular at least 20%, at least 50% orat least 100%. In one embodiment, the tumor-associated antigen isexpressed only in tissue of a diseased individual, while expression in ahealthy individual is repressed. One example of such a disease iscancer, wherein the term “cancer” according to the invention comprisesleukemias, seminomas, melanomas, teratomas, gliomas, kidney cancer,adrenal cancer, thyroid cancer, intestinal cancer, liver cancer, coloncancer, stomach cancer, gastrointestinal cancer, lymph node cancer,esophagus cancer, colorectal cancer, pancreas cancer, ear, nose andthroat (ENT) cancer, breast cancer, prostate cancer, cancer of theuterus, ovarian cancer and lung cancer and the matastases thereof.

According to the invention, a biological sample may be a tissue sampleand/or a cellular sample and may be obtained in the conventional mannersuch as by tissue biopsy, including punch biopsy, and by taking blood,bronchial aspirate, sputum, urine, feces or other body fluids, for usein the various methods described herein.

According to the invention, the term “immunoreactive cell” means a cellwhich can mature into an immune cell (such as B cell, T helper cell, orcytolytic T cell) with suitable stimulation. Immunoreactive cellscomprise CD34⁺ hematopoietic stem cells, immature and mature T cells andimmature and mature B cells. If production of cytolytic or T helpercells recognizing a tumor-associated antigen is desired, theimmunoreactive cell is contacted with a cell expressing atumor-associated antigen under conditions which favor production,differentiation and/or selection of cytolytic T cells and of T helpercells. The differentiation of T cell precursors into a cytolytic T cell,when exposed to an antigen, is similar to clonal selection of the immunesystem.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of antigen-presenting cells such ascancer cells which present one or more tumor-associated antigens. Inthis connection, for example autologous cytotoxic T lymphocytes specificfor a complex of a tumor-associated antigen and an MHC molecule areadministered to a patient having a cellular abnormality. The productionof such cytotoxic T lymphocytes in vitro is known. An example of amethod of differentiating T cells can be found in WO-A-9633265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes,fluorogenic tetramers of MHC class I molecule/peptide complexes are usedfor detecting specific clones of cytotoxic T lymphocytes (Altman et al.,Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).Soluble MHC class I molecules are folded in vitro in the presence of β₂microglobulin and a peptide antigen binding to said class I molecule.The MHC/peptide complexes are purified and then labeled with biotin.Tetramers are formed by mixing the biotinylated peptide-MHC complexeswith labeled avidin (e.g. phycoerythrin) in a molar ratio of 4:1.Tetramers are then contacted with cytotoxic T lymphocytes such asperipheral blood or lymph nodes. The tetramers bind to cytotoxic Tlymphocytes which recognize the peptide antigen/MHC class I complex.Cells which are bound to the tetramers may be sorted byfluorescence-controlled cell sorting to isolate reactive cytotoxic Tlymphocytes. The isolated cytotoxic T lymphocytes may then be propagatedin vitro.

In a therapeutic method referred to as adoptive transfer (Greenberg, J.Immunol. 136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynchet al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell59:603-614, 1989), cells presenting the desired complex (e.g. dendriticcells) are combined with cytotoxic T lymphocytes of the patient to betreated, resulting in a propagation of specific cytotoxic T lymphocytes.The propagated cytotoxic T lymphocytes are then administered to apatient having a cellular anomaly characterized by particular abnormalcells presenting the specific complex. The cytotoxic T lymphocytes thenlyse the abnormal cells, thereby achieving a desired therapeutic effect.

Often, of the T cell repertoire of a patient, only T cells with lowaffinity for a specific complex of this kind can be propagated, sincethose with high affinity have been extinguished due to development oftolerance. An alternative here may be a transfer of the T cell receptoritself. For this too, cells presenting the desired complex (e.g.dendritic cells) are combined with cytotoxic T lymphocytes of healthyindividuals or another species (e.g. mouse). This results in propagationof specific cytotoxic T lymphocytes with high affinity if the Tlymphocytes are derived from a donor organism which had no previouscontact with the specific complex. The high affinity T cell receptor ofthese propagated specific T lymphocytes is cloned. If the high affinityT cell receptors have been cloned from another species they can behumanized to a different extent. Such T cell receptors are thentransduced via gene transfer, for example using retroviral vectors, intoT cells of patients, as desired. Adoptive transfer is then carried outusing these genetically altered T lymphocytes (Stanislawski et al., NatImmunol. 2:962-70, 2001; Kessels et al., Nat Immunol. 2:957-61, 2001).

The therapeutic aspects above start out from the fact that at least someof the abnormal cells of the patient present a complex of atumor-associated antigen and an HLA molecule. Such cells may beidentified in a manner known per se. As soon as cells presenting thecomplex have been identified, they may be combined with a sample fromthe patient, which contains cytotoxic T lymphocytes. If the cytotoxic Tlymphocytes lyse the cells presenting the complex, it can be assumedthat a tumor-associated antigen is presented.

Adoptive transfer is not the only form of therapy which can be appliedaccording to the invention. Cytotoxic T lymphocytes may also begenerated in vivo in a manner known per se. One method usesnonproliferative cells expressing the complex. The cells used here willbe those which usually express the complex, such as irradiated tumorcells or cells transfected with one or both genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting HLA molecule). Various cell types may be used. Furthermore,it is possible to use vectors which carry one or both of the genes ofinterest. Particular preference is given to viral or bacterial vectors.For example, nucleic acids coding for a tumor-associated antigen or fora part thereof may be functionally linked to promoter and enhancersequences which control expression of said tumor-associated antigen or afragment thereof in particular tissues or cell types. The nucleic acidmay be incorporated into an expression vector. Expression vectors may benonmodified extrachromosomal nucleic acids, plasmids or viral genomesinto which exogenous nucleic acids may be inserted. Nucleic acids codingfor a tumor-associated antigen may also be inserted into a retroviralgenome, thereby enabling the nucleic acid to be integrated into thegenome of the target tissue or target cell. In these systems, amicroorganism such as vaccinia virus, pox virus, Herpes simplex virus,retrovirus or adenovirus carries the gene of interest and de facto“infects” host cells. Another preferred form is the introduction of thetumor-associated antigen in the form of recombinant RNA which may beintroduced into cells by liposomal transfer or by electroporation, forexample. The resulting cells present the complex of interest and arerecognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associatedantigen or a fragment thereof with an adjuvant in order to makeincorporation into antigen-presenting cells in vivo possible. Thetumor-associated antigen or a fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The tumor-associatedantigen is processed to produce a peptide partner for the HLA molecule,while a fragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toHLA molecules. Preference is given to administration forms in which thecomplete antigen is processed in vivo by a dendritic cell, since thismay also produce T helper cell responses which are needed for aneffective immune response (Ossendorp et al., Immunol Lett. 74:75-9,2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, itis possible to administer an effective amount of the tumor-associatedantigen to a patient by intradermal injection, for example. However,injection may also be carried out intranodally into a lymph node (Maloyet al., Proc Natl Acad Sci USA 98:3299-303, 2001). It may also becarried out in combination with reagents which facilitate uptake intodendritic cells. Preferred tumor-associated antigens comprise thosewhich react with allogenic cancer antisera or with T cells of manycancer patients. Of particular interest, however, are those againstwhich no spontaneous immune responses pre-exist. Evidently, it ispossible to induce against these immune responses which can lyse tumors(Keogh et al., J. Immunol. 167:787-96,. 2001; Appella et al., BiomedPept Proteins Nucleic Acids 1:177-84, 1995; Wentworth et al., MolImmunol. 32:603-12, 1995).

The pharmaceutical compositions described according to the invention mayalso be used as vaccines for immunization. According to the invention,the terms “immunization” or “vaccination” mean an increase in oractivation of an immune response to an antigen. It is possible to useanimal models for testing an immunizing effect on cancer by using atumor-associated antigen or a nucleic acid coding therefor. For example,human cancer cells may be introduced into a mouse to generate a tumor,and one or more nucleic acids coding for tumor-associated antigens maybe administered. The effect on the cancer cells (for example reductionin tumor size) may be measured as a measure for the effectiveness of animmunization by the nucleic acid.

As part of the composition for an immunization, one or moretumor-associated antigens or stimulating fragments thereof areadministered together with one or more adjuvants for inducing an immuneresponse or for increasing an immune response. An adjuvant is asubstance which is incorporated into the antigen or administeredtogether with the latter and which enhances the immune response.Adjuvants may enhance the immune response by providing an antigenreservoir (extracellularly or in macrophages), activating macrophagesand/or stimulating particular lymphocytes. Adjuvants are known andcomprise in a nonlimiting way monophosphoryl lipid A (MPL, SmithKlineBeecham), saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKlineBeecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells7:178-186, 1997), incomplete Freund's adjuvant, complete Freund'sadjuvant, vitamin E, montanide, alum, CpG oligonucleotides (cf. Kreig etal., Nature 374:546-9, 1995) and various water-in-oil emulsions preparedfrom biologically degradable oils such as squalene and/or tocopherol.Preferably, the peptides are administered in a mixture with DQS21/MPL.The ratio of DQS21 to MPL is typically about 1:10 to 10:1, preferablyabout 1:5 to 5:1 and in particular about 1:1. For administration tohumans, a vaccine formulation typically contains DQS21 and MPL in arange from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids. Examples of such costimulating molecules are B7-1 and B7-2 (CD80and CD86, respectively) which are expressed on dendritic cells (DC) andinteract with the CD28 molecule expressed on the T cells. Thisinteraction provides a costimulation (signal 2) for anantigen/MHC/TCR-stimulated (signal 1) T cell, thereby enhancingpropagation of said T cell and the effector function. B7 also interactswith CTLA4 (CD152) on T cells, and studies involving CTLA4 and B7ligands demonstrate that B7-CTLA4 interaction can enhance antitumorimmunity and CTL propagation (Zheng, P. et al., Proc. Natl. Acad. Sci.USA 95(11):6284-6289 (1998)).

B7 is typically not expressed on tumor cells so that these are noeffective antigen-presenting cells (APCs) for T cells. Induction of B7expression would enable tumor cells to stimulate more effectivelypropagation of cytotoxic T lymphocytes and an effector function.Costimulation by a combination of B7/IL-6/IL-12 revealed induction ofIFN-gamma and Th1-cytokine profile in a T cell population, resulting infurther enhanced T cell activity (Gajewski et al., J. Immunol.154:5637-5648 (1995)).

A complete activation of cytotoxic T lymphocytes and a complete effectorfunction require an involvement of T helper cells via interactionbetween the CD40 ligand on said T helper cells and the CD40 moleculeexpressed by dendritic cells (Ridge et al., Nature 393:474 (1998),Bennett et al., Nature 393:478 (1998), Schönberger et al., Nature393:480 (1998)). The mechanism of this costimulating signal probablyrelates to the increase in B7 production and associated IL-6/IL-12production by said dendritic cells (antigen-presenting cells).CD40-CD40L interaction thus complements the interaction of signal 1(antigen/MHC-TCR) and signal 2 (B7-CD28).

The use of anti-CD40 antibodies for stimulating dendritic cells would beexpected to directly enhance a response to tumor antigens which areusually outside the range of an inflammatory response or which arepresented by nonprofessional antigen-presenting cells (tumor cells). Inthese situations, T helper and B7-costimulating signals are notprovided. This mechanism could be used in connection with therapiesbased on antigen-pulsed dendritic cells.

The invention also provides for administration of nucleic acids,polypeptides or peptides. Polypeptides and peptides may be administeredin a manner known per se. In one embodiment, nucleic acids areadministered by ex vivo methods, i.e. by removing cells from a patient,genetic modification of said cells in order to incorporate atumor-associated antigen and reintroduction of the altered cells intothe patient. This generally comprises introducing a functional copy of agene into the cells of a patient in vitro and reintroducing thegenetically altered cells into the patient. The functional copy of thegene is under the functional control of regulatory elements which allowthe gene to be expressed in the genetically altered cells. Transfectionand transduction methods are known to the skilled worker. The inventionalso provides for administering nucleic acids in vivo by using vectorssuch as viruses and target-controlled liposomes.

In a preferred embodiment, a viral vector for administering a nucleicacid coding for a tumor-associated antigen is selected from the groupconsisting of adenoviruses, adeno-associated viruses, pox viruses,including vaccinia virus and attenuated pox viruses, Semliki Forestvirus, retroviruses, Sindbis virus and Ty virus-like particles.Particular preference is given to adenoviruses and retroviruses. Theretroviruses are typically replication-deficient (i.e. they areincapable of generating infectious particles).

Various methods may be used in order to introduce according to theinvention nucleic acids into cells in vitro or in vivo. Methods of thiskind comprise transfection of nucleic acid CaPO₄ precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection, and the like. In particular embodiments,preference is given to directing the nucleic acid to particular cells.In such embodiments, a carrier used for administering a nucleic acid toa cell (e.g. a retrovirus or a liposome) may have a bound target controlmolecule. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell may be incorporated into or attached to the nucleicacid carrier. Preferred antibodies comprise antibodies which bindselectively a tumor-associated antigen. If administration of a nucleicacid via liposomes is desired, proteins binding to a surface membraneprotein associated with endocytosis may be incorporated into theliposome formulation in order to make target control and/or uptakepossible. Such proteins comprise capsid proteins or fragments thereofwhich are specific for a particular cell type, antibodies to proteinswhich are internalized, proteins addressing an intracellular site, andthe like.

The therapeutic compositions of the invention may be administered inpharmaceutically compatible preparations. Such preparations may usuallycontain pharmaceutically compatible concentrations of salts, buffersubstances, preservatives, carriers, supplementing immunity-enhancingsubstances such as adjuvants, CpG and cytokines and, where appropriate,other therapeutically active compounds.

The therapeutically active compounds of the invention may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitonealy, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lung aerosol. Antisense nucleic acids are preferablyadministered by slow intravenous administration.

The compositions of the invention are administered in effective amounts.An “effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of a particularcondition characterized by expression of one or more tumor-associatedantigens, the desired reaction relates to inhibition of the course ofthe disease. This comprises slowing down the progress of the diseaseand, in particular, interrupting the progress of the disease. Thedesired reaction in a treatment of a disease or of a condition may alsobe delay of the onset or a prevention of the onset of said disease orsaid condition.

An effective amount of a composition of the invention will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect.

The doses administered of the compositions of the invention may dependon various parameters such as the type of administration, the conditionof the patient, the desired period of administration, etc. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg,preferably from 10 ng to 100 μg, are formulated and administered for atreatment or for generating or increasing an immune response. If theadministration of nucleic acids (DNA and RNA) coding fortumor-associated antigens is desired, doses of from 1 ng to 0.1 mg areformulated and administered.

The pharmaceutical compositions of the invention are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible compositions. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier. According to the invention, theterm “pharmaceutically compatible carrier” refers to one or morecompatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to humans. The term“carrier” refers to an organic or inorganic component, of a natural orsynthetic nature, in which the active component is combined in order tofacilitate application. The components of the pharmaceutical compositionof the invention are usually such that no interaction occurs whichsubstantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the invention may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, suspensions, syrups, elixir or in the form of an emulsion, forexample.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting.

Owing to the description and the examples, further embodiments which arelikewise included in the invention are accessible to the skilled worker.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. GPR35 mRNA expression in colon carcinoma biopsies

RT-PCR investigations with DNA-free RNA show GPR35 expression in most ofthe colon carcinoma biopsies. By contrast, there is no detectableexpression in normal tissues. (1-Breast, 2-lung, 3-lymph nodes,4-thymus, 5-colon, 6-15 colon carcinoma, 16-neg. control).

FIG. 2. Quantitative PCR analysis of GUCY2C mRNA expression in normaland tumor tissues

Real-time PCR investigation with GUCY2C-specific primers (SEQ ID NO:22-23) shows selective mRNA expression in normal ileum, colon, and inall colon carcinoma biopsies. Distinct quanities of GUCY2C transcriptswere also detected in a colon carcinoma metastasis in the liver.

FIG. 3. Identification of tumor-specific GUCY2C splice variants

PCR products from normal colon tissues and colon carcinomas were cloned,and clones from both groups were checked by restriction analysis (EcoRI) and sequenced.

FIG. 4. Selective SCGB3A expression in normal lung and lung carcinoma

RT-PCR analysis with gene-specific SCGB3A2 primers (SEQ ID NO: 37, 38)shows cDNA amplification exclusively in normal lung (lane 8, 14-15) andin lung carcinoma biopsies (lane 16-24). (1-Liver-N, 2-PBMC-N, 3-lymphnode-N, 4-stomach-N, 5-testis-N, 6-breast-N, 7-kidney-N, 8-lung-N,9-thymus-N, 10-ovary-N, 11-adrenal-N, 12-spleen-N, 14-15-lung-N,16-24-lung carcinoma, 25-negative control).

FIG. 5. Claudin-18A2.1 expression in stomach, esophagus, stomachcarcinoma and pancreatic carcinoma

RT-PCR analysis with claudin-18A2.1-specific primers (SEQ ID NO: 39, 40)showed according to the invention pronounced claudin-18A2.1 expressionin 8/10 stomach carcinoma biopsies and in 3/6 pancreatic carcinomabiopsies. Distinct expression was also detected in stomach and normalesophageal tissue. In contrast thereto, no expression was detected inthe ovary and in ovarian carcinoma.

FIG. 6. SLC13A1 expression in the kidney and renal cell carcinoma

RT-PCR analysis with SLC13A1-specific primers (SEQ ID NO: 49, 50) showedexpression in 7/8 renal cell carcinoma samples. Otherwise, transcriptswithin normal tissues were detected exclusively in the kidney.(1-2-kidney, 3-10-renal cell carcinoma, 11-breast, 12-lung, 13-liver,14-colon, 15-lymph nodes, 16-spleen, 17-esophagus, 18-thymus,19-thyroid, 20-PBMCs, 21-ovary, 22-testis).

FIG. 7. CLCA1 expression in colon, colon carcinoma and stomach carcinoma

RT-PCR investigations with CLCA1-specific primers. (SEQ ID NO: 67, 68)confirmed selective expression in the colon and showed high expressionin (3/7) investigated colon carcinoma and (1/3) investigated stomachcarcinoma samples. The other normal tissues (NT) showed no or only veryweak expression.

FIG. 8. FLJ21477 expression in the colon and colon carcinoma

RT-PCR investigations with FLJ21477-specific primers (SEQ ID NO: 69, 70)showed selective expression in the colon and additionally various levelsof expression in (7/12) investigated colon carcinoma samples. The othernormal tissues (NT) showed no expression.

FIG. 9. FLJ20694 expression in the colon and colon carcinoma

RT-PCR investigations with FLJ20694-specific primers (SEQ ID NO: 71, 72)showed selective expression in the colon and additionally various levelsof expression in (5/9) investigated colon carcinoma samples. The othernormal tissues (NT) showed no expression.

FIG. 10. von Ebner expression in stomach, lung and lung carcinoma

RT-PCR investigations with von Ebner-specific primers (SEQ ID NO: 73,74) showed selective expression in the stomach, in the lung and in(5/10) investigated lung carcinoma samples. The other normal tissues(NT) showed no expression.

FIG. 11. Plunc expression in thymus, lung and lung carcinoma

RT-PCR investigations with Plunc-specific primers (SEQ ID NO: 75, 76)showed selective expression in the thymus, in the lung and in (6/10)investigated lung carcinoma samples. The other normal tissues showed noexpression.

FIG. 12. SLC26A9 expression in lung, lung carcinoma and thyroid

RT-PCR investigations with SLC26A9-specific primers (SEQ ID NO: 77, 78)showed selective expression in the lung and in all (13/13) investigatedlung carcinoma samples. The other normal tissues (NT) showed noexpression with the exception of the thyroid.

FIG. 13. THC1005163 expression in stomach, ovary, lung and lungcarcinoma

RT-PCR investigations with a THC1005163-specific primer (SEQ ID NO: 79)and a nonspecific oligo dT tag primer showed expression in stomach,ovary, lung and in (5/9) lung carcinoma biopsies. The other normaltissues (NT) showed no expression.

FIG. 14. LOC134288 expression in kidney and renal cell carcinoma

RT-PCR investigations with LOC134288-specific primers (SEQ ID NO: 80,81) showed selective expression in the kidney and in (5/8) investigatedrenal cell carcinoma biopsies.

FIG. 15. THC943866 expression in kidney and renal cell carcinoma

RT-PCR investigations with THC943866-specific primers (SEQ ID NO: 82,83) showed selective expression in the kidney and in (4/8) investigatedrenal cell carcinoma biopsies.

FIG. 16. FLJ21458 expression in colon and colon carcinoma

RT-PCR investigations with FLJ21458-specific primers (SEQ ID NO: 86, 87)showed selective expression in the colon and in (7/10) investigatedcolon carcinoma biopsies. (1-2-colon, 3-liver, 4-PBMCs, 5-spleen,6-prostate, 7-kidney, 8-ovary, 9-skin, 10-ileum, 11-lung, 12-testis,13-22 colon carcinoma, 23-neg. control).

FIG. 17. Cellular localization of GPR35

Immunofluorescence for detecting the cellular localization of GPR35after transfection of a plasmid that expresses a GPR35-GFP fusionprotein. The arrows identify the membrane-associated fluorescence of thefluorescent GFP.

FIG. 18. Quantitative expression of GPR35

-   -   A. Quantitative RT-PCR with GPR35-specific primers (SEQ ID NO:        88, 89) show selective expression in the intestine, in colon        tumor samples and in metastases from intestinal tumors. The        following normal tissues were analyzed: liver, lung, lymph        nodes, stomach, spleen, adrenal, kidney, esophagus, ovary,        testis, thymus, skin, breast, pancreas, lymphocytes, activated        lymphocytes, prostate, thyroid, fallopian tube, endometrium,        cerebellum, brain.    -   B. Prevalence of GPR35 in colon tumors and metastases thereof.        GPR35 is expressed both in the tumor and in metastases in more        than 90% of the cases.

FIG. 19. Quantitative expression of GUCY2C

Quantitative RT-PCR with GUCY2C-specific primers (SEQ ID NO: 98, 99)show high and selective expression in normal colonic and gastric tissue(A) and GUCY2C-specific expression in colonic and gastric tumor samples(B). GUCY2C is detectable in 11/12 colon carcinomas and in 7/10 stomachcarcinomas.

FIG. 20. Quantitative expression of SCGB3A2

Quantitative RT-PCR with SCGB3A2-specific primers (SEQ ID NO: 103, 104)show selective expression in lung samples and lung tumor samples. 19/20lung tumor samples are SCGB3A2-positive, and SCGB3A2 is over-expressedby a factor of at least 10 in more than 50% of the samples. Thefollowing normal tissues were analyzed: liver, lung, lymph nodes,stomach, spleen, adrenal, kidney, esophagus, ovary, testis, thymus,skin, breast, pancreas, lymphocytes, activated lymphocytes, prostate,thyroid, fallopian tube, endometrium, cerebellum, brain.

FIG. 21. Immunofluorescence with SCGB3A2-specific anti-bodies

COS7 cells were transfected with a plasmid which codes for anSCGB3A2-GFP fusion protein. A. Detection of the transfected fusionprotein with an SCGB3A2-specific rabbit antiserum (immunization with SEQID NO: 105). B. Detection of the transfected fusion protein by GFPfluorescence. C. Superimposition of the two fluorescences from A and B.The yellow color is produced at the points where the two fluorescencesare superimposed and thus demonstrates the specificity of the SCGB3A2antiserum.

FIG. 22. Diagrammatic depiction of claudin-18 splice variants

The two claudin-18 splice variants A1 and A2 differ in the N terminusand show different potential glycosylation sites.

FIG. 23. Quantitative expression of claudin-18, variant A1

Claudin-A1 is highly activated in a large number of tumor tissues.Particularly strong expression is found in gastric tumors, lung tumors,pancreatic carcinomas and esophageal carcinomas.

FIG. 24. Quantitative expression of claudin-18, variant A2

Variant A2 is, like variant A1, activated in many tumors.

FIG. 25. Use of claudin-18A2-specific antibodies (extracellular domain)

(Top) Staining of claudin-18A2-positive gastric carcinoma cells (SNU-16)with an antibody which was produced by immunization with a peptide (SEQID NO: 17). Membrane staining appears particularly strongly in thecell/cell interaction regions. A-preimmune, MeOH; B-immune serum MeOH, 5μg/ml; (Below) Demonstration of the specificity of the antibody bycolocalization analysis in claudin-18A2-GFP-transfected 293 T cells.A-Claudin-18A2 GFP; B-anti-claudin-A2; C-superimposition.

FIG. 26. Use of claudin-18A2-specific antibodies (extracellular domain)

Membrane staining of claudin-18A2-positive gastric carcinoma cells(SNU-16) with an antibody which was produced by immunization with apeptide (SEQ ID NO: 113, N-terminally located extracellular domain). Amonoclonal antibody which is directed against E-cadherin was used forcounterstaining. A-antibody; B-counterstaining; C-superimposition.

FIG. 27. Use of antibodies against the C-terminal extracellular domainof claudin-18

(Left, top and below) Membrane staining of claudin-18A2-positive gastriccarcinoma cells (SNU-16) with an antibody which was produced byimmunization with a peptide (SEQ ID NO: 116, C-terminally locatedextra-cellular domain). A monoclonal antibody which is directed againstE-cadherin was used for counter-staining (right top, below).

FIG. 28. Use of claudin-18A1-specific antibodies

(Top) Weak to absent staining of gastric carcinoma cells (SNU-16;claudin18A2 positive) with an antibody which was produced byimmunization with a claudin-18A1-specific peptide (SEQ ID NO: 115).A-anti-E-cadherin; B-anti-claudin-18A1; C-superimposition. (Below)Demonstration of the specificity of the antibody by colocalizationanalysis in claudin-18A1-GFP-transfected 293T cells. A-GFP-claudin-18A1;B-anti-claudin-18A1; C-superimposition.

FIG. 29. Detection of claudin-18A2 in a Western blot.

Western blotting with lysates from various healthy tissues with aclaudin-18A2-specific antibody directed against the epitope with SEQ IDNO: 17. 1-Stomach; 2-testis; 3-skin; 4-breast; 5-liver; 6-colon; 7-lung;8-kidney; 9-lymph nodes.

FIG. 30. Claudin-18A2 Western blotting with samples from stomach andstomach tumors

Lysates from stomach and stomach tumors were blotted and tested using aclaudin-18A2-specific antibody against the epitope having SEQ ID NO: 17.Stomach tumors show a less glycosylated form of claudin-18A2. PNGase Ftreatment of stomach lysates leads to the formation of thelow-glycosylated form.

-   Left: 1-stomach No #A; 2-stomach Tu #A; 3-stomach No #B; 4-stomach    Tu #B-   Right: 1-stomach No #A; 2-stomach No #B; 3-stomach No #B+PNGase F;    4-stomach Tu #C; 5-stomach Tu #D; 6-stomach Tu #D+PNGase F

FIG. 31. Expression of claudin-18 in lung tumors

Low-glycosylated claudin-18A2 variants were detected in lung tumors inaccordance with FIG. 30. 1-Stomach No; 2-stomach Tu; 3-9-lung Tu.

FIG. 32. Immunohistochemical analysis of claudin-18 usingclaudin-18A2-specific antibodies in stomach tumor tissue

FIG. 33. Indirect immunofluorescence of stomach-specific Snu16 cellswith a claudin-18-specific polyclonal antiserum

A. Staining with a preimmune serum generated before the immunization; B.Staining with the claudin-18-specific serum.

FIG. 34. Quantitative expression of SLC13A1

Quantitative RT-PCR with SLC13A1-specific primers (SEQ ID NO: 121, 122)show high and selective expression in normal kidney tissue (A) andSLC13A1-specific expression in renal cell carcinomas (B). SLC13A1transcription is detectable in 5/8 renal cell carcinomas.

FIG. 35. Cellular localization of SLC13A1

Immunofluorescence to demonstrate the cellular localization of SLC13A1after transfection of a plasmid which provides an SLC13A1-GFP fusionprotein. The membrane-associated fluorescence of the SLC13A1 fusionprotein is to be seen clearly (as ring around the transfected cell).

FIG. 36. Quantitative expression of CLCA1

Quantitative RT-PCR with CLCA1-specific primers (SEQ ID NO: 125, 126)show high and selective expression in normal colonic tissue and stomachtissue (A) and CLCA1-specific expression in colonic and gastric tumorsamples (B). CLCA1 is detectable in 6/12 colon carcinomas and in 7/10stomach carcinomas.

FIG. 37. Quantitative expression of FLJ21477

Quantitative RT-PCR with FLJ21477-specific primers (SEQ ID NO: 127, 128)show high and selective expression in normal colonic and gastric tissueand weak expression in thymus, esophagus and brain (A) and theFLJ21477-specific expression in colonic tumor samples (B). FLJ21477 isdetectable in 11/12 colon carcinomas.

FIG. 38. Quantitative expression of FLJ20694

Quantitative RT-PCR with FLJ20694-specific primers (SEQ ID NO: 129, 130)show high and selective expression in normal colonic and gastric tissue(A) and FLJ20694-specific overexpression in colonic and gastric tumorsamples (B). FLJ20694 is detectable in 11/12 colon carcinomas and in7/10 stomach carcinomas.

FIG. 39. Quantitative expression of FLJ21458

Quantitative RT-PCR with FLJ21458-specific primers (SEQ ID NO: 133, 134)show selective expression in testis, gastric and intestinal tissue. Inaddition, FLJ21458-specific transcripts were detectable in 20/20 colonictumors and in 7/11 colonic metastases. The following normal tissues wereanalyzed: liver, lung, lymph nodes, spleen, adrenal, kidney, esophagus,ovary, testis, thymus, skin, breast, pancreas, lymphocytes, activatedlymphocytes, prostate, thyroid, fallopian tube, endometrium, cerebellum,brain.

FIG. 40. Immunofluorescence with FLJ21458-specific antibodies

(Top) 293 cells were transfected with a plasmid which codes for anFLJ21458-GFP fusion protein. A: detection of the transfected fusionprotein with an FLJ21458-specific rabbit antiserum (immunization withSEQ ID NO: 136). B: detection of the transfected fusion protein by GFPfluorescence. C: superimposition of the two fluorescences from A and B.The yellow color is produced at the points where the two fluorescencesare superimposed and thus demonstrates the specificity of the FLJ21458antiserum.

(Below) Analysis of Snu16 cells which endogenously synthesize FLJ21458.A: protein detection using an FLJ21458-specific rabbit antiserum(immunization with SEQ ID NO: 136). B: detection of the membrane proteinE-cadherin. C: superimposition of the two fluorescences from A and B.The yellow color is produced at the points where the two fluorescencesare superimposed, and demonstrates the membrane localization ofFLJ21458.

FIG. 41. Sequences

The sequences to which reference is made herein are shown.

EXAMPLES Material and Methods

The terms “in silico”, “electronic” and “virtual cloning” refer solelyto the utilization of methods based on databases, which may also be usedto simulate laboratory experimental processes.

Unless expressly defined otherwise, all other terms and expressions areused so as to be understood by the skilled worker. The techniques andmethods mentioned are carried out in a manner known per se and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. All methods including the use of kitsand reagents are carried out according to the manufacturers'information.

Datamining-based Strategy for Determining New Tumor-associated Genes

Two in silico strategies, namely GenBank keyword search and thecDNAxProfiler, were combined. Utilizing the NCBI ENTREZ Search andRetrieval System (http://www.ncbi.nlm.nih.gov/Entrez), a GenBank searchwas carried out for candidate genes annotated as being specificallyexpressed in specific tissues (Wheeler et al., Nucleic Acids Research28:10-14, 2000).

Carrying out queries with keywords such as “colon-specific gene”,“stomach-specific gene” or “kidney-specific gene”, candidate genes (GOI,genes of interest) were extracted from the databases. The search wasrestricted to part of the total information of these databases by usingthe limits “homo sapiens”, for the organism, and “mRNA”, for the type ofmolecule.

The list of the GOI found was curated by determining different names forthe same sequence and eliminating such redundancies.

All candidate genes obtained by the keyword search were in turn studiedwith respect to their tissue distribution by the “electronic Northern”(eNorthen) method. The eNorthern is based on aligning the sequence of aGOI with an EST (expressed sequence tag) database (Adams et al., Science252:1651, 1991) (http://www.ncbi.nlm.nih.gov/BLAST). The tissue originof each EST which is found to be homologous to the inserted GOI can bedetermined and in this way the sum of all ESTs produces a preliminaryassessment of the tissue distribution of the GOI. Further studies werecarried out only with those GOI which had no homologies to EST from nonorgan-specific normal tissues. This evaluation also took into accountthat the public domain contains wrongly annotated cDNA libraries(Scheurle et al., Cancer Res. 60:4037-4043, 2000)(www.fau.edu/cmbb/publications/cancergenes6.htm).

The second datamining method utilized was the cDNA xprofiler of the NCBICancer Genome Anatomy Project(http://cgap.nci.nih.gov/Tissues/xProfiler) (Hillier et al., GenomeResearch 6:807-828, 1996; Pennisi, Science 276:1023-1024, 1997). Thisallows pools of transcriptomes deposited in databases to be related toone another by logical operators. We have defined a pool A to which allexpression libraries prepared for example from colon were assigned,excluding mixed libraries. All cDNA libraries prepared from normaltissues other than colon were assigned to pool B. Generally, all cDNAlibraries were utilized independently of underlying preparation methods,but only those with a size>1000 were admitted. Pool B was digitallysubtracted from pool A by means of the BUT NOT operator. The set of GOIfound in this manner was also subjected to eNorthern studies andvalidated by a literature research.

This combined datamining includes all of the about 13,000 full-lengthgenes in the public domain and predicts out of these genes havingpotential organ-specific expression.

All other genes were first evaluated in normal tissues by means ofspecific RT-PCR. All GOI which had proved to be expressed in non-organspecific normal tissues had to be regarded as false-positives and wereexcluded from further studies. The remaining ones were studied in alarge panel of a wide variety of tumor tissues. The antigens depictedbelow proved here to be activated in tumor cells.

RNA Extraction, Preparation of poly-d(T) Primed cDNA and ConventionalRT-PCR Analysis

Total RNA was extracted from native tissue material by using guanidiumisothiocyanate as chaotropic agent (Chomczynski & Sacchi, Anal. Biochem.162:156-9, 1987). After extraction with acidic phenol and precipitationwith isopropanol, said RNA was dissolved in DEPC-treated water.

First strand cDNA synthesis from 2-4 μg of total RNA was carried out ina 20 μl reaction mixture by means of Superscript II (Invitrogen),according to the manufacturer's information. The primer used was adT(18) oligonucleotide. Integrity and quality of the cDNA were checkedby amplification of p53 in a 30 cycle PGR (sense CGTGAGCGCTTCGAGATGTTCCG(SEQ ID NO. 138), antisense CCTAACCAGCTGCCCAACTGTAG (SEQ ID NO. 139),hybridization temperature 67 ° C.).

An archive of first strand cDNA was prepared from a number of normaltissues and tumor entities. For expression studies, 0.5 μl of thesecDNAs was amplified in a 30 μl reaction mixture, using GOI-specificprimers (see below) and 1 U of HotStarTaq DNA polymerase (Qiagen). Eachreaction mixture contained 0.3 mM dNTPs, 0.3 μM of each primer and 3 μlof 10× reaction buffer. The primers were selected so as to be located intwo different exons, and elimination of the interference bycontaminating genomic DNA as the reason for false-positive results wasconfirmed by testing nonreverse-transcribed DNA as template. After 15minutes at 95° C. to activate the HotStarTaq DNA polymerase, 35 cyclesof PCR were carried out (1 min at 94° C., 1 min at the particularhybridization temperature, 2 min at 72° C. and final elongation at 72°C. for 6 min). 20 μl of this reaction were fractionated and analyzed onan ethidium bromide-stained agarose gel.

The following primers were used for expression analysis of thecorresponding antigens at the hybridization temperature indicated.

GPR35 (65° C.) Sense: 5′-AGGTACATGAGCATCAGCCTG-3′ (SEQ ID NO. 20)Antisense: 5′-GCAGCAGTTGGCATCTGAGAG-3′ (SEQ ID NO. 21) GUCY2C (62° C.)Sense: 5′-GCAATAGACATTGCCAAGATG-3′ (SEQ ID NO. 22) Antisense:5′-AACGCTGTTGATTCTCCACAG-3′ (SEQ ID NO. 23) SCGB3A2 (66° C.) Sense:5′-CAGCCTTTGTAGTTACTCTGC-3′ (SEQ ID NO. 37) Antisense:5′-TGTCACACCAAGTGTGATAGC-3′ (SEQ ID NO. 38) Claudin18A2 (68° C.) Sense1:5′-GGTTCGTGGTTTCACTGATTGGGATTGC-3′ (SEQ ID NO. 39) Antisense1:5′-CGGCTTTGTAGTTGGTTTCTTCTGGTG-3′ (SEQ ID NO. 40) Sense2:5′-TGTTTTCAACTACCAGGGGC-3′ (SEQ ID NO. 107) Antisense2:5′-TGTTGGCTTTGGCAGAGTCC-3′ (SEQ ID NO. 108) Claudin18A1 (64° C.) Sense:5′-GAGGCAGAGTTCAGGCTTCACCGA-3′ (SEQ ID NO. 109) Antisense:5′-TGTTGGCTTTGGCAGAGTCC-3′ (SEQ ID NO. 110) SLCI3A1 (64° C.) Sense:5′-CAGATGGTTGTGAGGAGTCTG-3′ (SEQ ID NO. 50) Antisense:5′-CCAGCTTTAACCATGTCAATG-3′ (SEQ ID NO. 49) CLCA1 (62° C.) Sense:5′-ACACGAATGGTAGATACAGTG-3′ (SEQ ID NO. 67) Antisense:5′-ATACTTGTGAGCTGTTCCATG-3′ (SEQ ID NO. 68) FLJ21477 (68° C.) Sense:5′-ACTGTTACCTTGCATGGACTG-3′ (SEQ ID NO. 69) Antisense:5′-CAATGAGAACACATGGACATG-3′ (SEQ ID NO. 70) FLJ20694 (64° C.) Sense:5′-CCATGAAAGCTCCATGTCTA-3′ (SEQ ID NO. 140) Antisense:5′-AGAGATGGCACATATTCTGTC (SEQ ID NO. 72) Ebner (70° C.) Sense:5′-ATCGGCTGAAGTCAAGCATCG-3′ (SEQ ID NO. 73) Antisense:5′-TGGTCAGTGAGGACTCAGCTC-3′ (SEQ ID NO. 74) Plunc (55° C.) Sense:5′-TTTCTCTGCTTGATGCACTTG-3′ (SEQ ID NO. 75) Antisense:5′-GTGAGCACTGGGAAGCAGCTC-3′ (SEQ ID NO. 76) SLC26A9 (67° C.) Sense:5′-GGCAAATGCTAGAGACGTGA-3′ (SEQ ID NO. 141) Antisense:5′-AGGTGTCCTTCAGCTGCCAAG-3′ (SEQ ID NO. 78) THC1005163 (60° C.) Sense:5′-GTTAAGTGCTCTCTGGATTTG-3′ (SEQ ID NO. 79) LOC134288 (64° C.) Sense:5′-ATCCTGATTGCTGTGTGCAAG-3′ (SEQ ID NO. 80) Antisense:5′-CTCTTCTAGCTGGTCAACATC-3′ (SEQ ID NO. 81) THC943866 (59° C.) Sense:5′-CCAGCAACAACTTACGTGGTC-3′ (SEQ ID NO. 82) Antisense:5′-CCTTTATTCACCCAATCACTC-3′ (SEQ ID NO. 83) FLJ21458 (62° C.) Sense:5′-ATTCATGGTTCCAGCAGGGAC-3′ (SEQ ID NO. 86) Antisense:5′-GGGAGACAAAGTCACGTACTC-3′ (SEQ ID NO. 87)

Preparation of Random Hexamer-primed cDNA and Quantitative Real-time PCR

The expression of several genes was quantified by real-time PCR. The PCRproducts were detected using SYBR Green as intercalating reporter dye.The reporter fluorescence of SYBR Green is suppressed in solution andthe dye is active only after binding to double-stranded DNA fragments.The increase in the SYBR Green fluorescence as a result of the specificamplification using GOI-specific primers after each PCR cycle isutilized for quantification. Expression of the target gene is quantifiedabsolutely or relative to the expression of a control gene with constantexpression in the tissues to be investigated. Expression was measuredafter standardization of the samples against 18 s RNA as so-calledhousekeeping gene using the ΔΔ-C_(t) method (PE Biosystems, USA). Thereactions were carried out in duplicates and determined in triplicates.The QuantiTect SYBR Green PCR kit (Qiagen, Hilden) was used inaccordance with the manufacturer's instructions. The cDNA wassynthesized using the high capacity cDNA Archive Kit (PE Biosystems,USA) with use of hexamer primers in accordance with the manufacturer'sinstructions. Each 5 μl portions of the diluted cDNA were employed in atotal volume of 25 μl for the PCR: sense primer 300 nM, antisense primer300 nM; initial denaturation 95° C. for 15 min; 95° C. for 30 sec;annealing for 30 sec; 72° C. for 30 sec; 40 cycles. The sequences of theprimers used are indicated in the respective examples.

Cloning and Sequence Analysis

Cloning of full-lengths and gene fragments took place by conventionalmethods. To ascertain the sequence, corresponding antigenes wereamplified using the proofreading polymerase pfu (Stratagene). Aftercompletion of the PCR, adenosine was ligated by means of HotStarTaq DNApolymerase to the ends of the amplicon in order to clone the fragmentsin accordance with the manufacturer's instructions into the TOPO-TAvector. The sequencing was carried out by a commercial service. Thesequences were analysed using conventional prediction programs andalgorithms.

Western Blotting

Cells from cell culture (endogenous expression of the target gene orsynthesis of the target protein after transfection of an expressionvector which encodes the target protein) or tissue samples which mightcontain the target protein are lysed in a 1% SDS solution. The SDSdenatures the proteins present in the lysate. The lysates of anexperimental mixture are fractionated according to size byelectrophoresis on 8-15% denaturing polyacrylamide gels (containing 1%SDS) depending on the expected protein size (SDS polyacrylamide gelelectrophoresis, SDS-PAGE). The proteins are then transferred by thesemi-dry electroblotting method (Biorad) to nitrocellulose membrane(Schleicher & Schüll) on which the desired protein can be detected. Forthis purpose, the membrane is initially blocked (e.g. with milk powder)and then incubated with the specific antibody in a dilution of1:20-1:200 (depending on the specificity of the antibody) for 60minutes. After a washing step, the membrane is incubated with a secondantibody coupled to a marker (e.g. enzymes such as peroxidase oralkaline phosphatase) which recognizes the first antibody. After afurther washing step, subsequently the target protein is visualized in acolor or chemiluminescence reaction on the membrane by means of anenzyme reaction (e.g. ECL, Amersham Bioscience). The result isdocumented by photographing with a suitable camera.

Analysis of protein modifications usually takes place by Westernblotting. Glycosilations, which usually have a size of several kDa, leadto a larger total mass of the target protein, which can be fractionatedin the SDS-PAGE. To detect specific O- and N-glycosidic linkages,protein lysates from tissues or cells are incubated before denaturationby SDS with O- or N-glycosidases (in accordance with their respectivemanufacturer's instructions, e.g. PNgase, endoglycosidase F,endoglycosidase H, Roche Diagnostics). This is followed by Westernblotting as described above. Thus, if there is a reduction in the sizeof a target protein after incubation with a glycosidase it is possibleto detect a specific glycosilation and, in this way, also analyse thetumor specificity of a modification. The exact position of theglycosilated amino acid can be predicted with algorithms and predictionprograms.

Immunofluorescence

Cells of established cell lines which either synthesize the targetprotein endogenously (detection of the RNA in RT-PCR or of the proteinby Western blotting) or else have been transfected with plasmid DNAbefore the IF are used. A wide variety of methods (e.g. electroporation,liposome-based transfection, calcium phosphate precipitation) are wellestablished for transfecting cell lines with DNA (e.g. Lemoine et al.Methods Mol. Biol. 1997; 75: 441-7). The transfected plasmid may in theimmunofluorescence encode the unmodified protein or else couple variousamino acid markers to the target protein. The most important markersare, for example, the fluorescing “green fluorescent protein” (GFP) inits various differentially fluorescing forms and short peptide sequencesof 6-12 amino acids for which high-affinity and specific antibodies areavailable. Cells which synthesize the target protein are fixed withparaformaldehyde, saponin or methanol. The cells can then if required bepermeabilized by incubation with detergents (e.g. 0.2% Triton X-100).After the fixation/permeabilization, the cells are incubated with aprimary antibody which is directed against the target protein or againstone of the coupled markers. After a washing step, the mixture isincubated with a second antibody coupled to a fluorescent marker (e.g.fluorescin, Texas Red, Dako) which binds to the first antibody. Thecells labeled in this way are then covered with a layer of glycerol andanalysed with the aid of a fluorescence microscope according to themanufacturer's instructions. Specific fluorescence emissions areachieved in this case by specific excitation depending on the substancesemployed. The analysis normally allows reliable localization of thetarget protein, the antibody quality and the target protein beingconfirmed in double stainings to stain in addition to the target proteinalso the coupled amino acid markers or other marker proteins whoselocalization has been described in the literature. GFP and itsderivatives represents a special case that can be directly excited anditself fluoresces, so that no antibodies are necessary for thedetection.

Immunohistochemistry

IHC serves specifically for (1) being able to estimate the amount oftarget protein in tumor and normal tissues, (2) analysing how many cellsin the tumor and healthy tissue synthesize the target gene, and/or (3)defining the cell type in a tissue (tumor, healthy cells) in which thetarget protein is detectable. Different protocols must be used dependingon the individual antibody (e.g. “Diagnostic Immunohistochemistry byDavid J., MD Dabbs ISBN: 0443065667” or in “Microscopy,Immunohistochemistry, and Antigen Retrieval Methods: For Light andElectron Microscopy ISBN: 0306467704”).

Immunohistochemistry (IHC) on specific tissue samples serves to detectprotein in the corresponding tissue. The aim of this method is toidentify the localization of a protein in a functionally intact tissueaggregate.

IHC serves specifically for (1) being able to estimate the amount oftarget protein in tumor and normal tissues, (2) analysing how many cellsin tumor and healthy tissue synthesize the target gene, and (3) definingthe cell type in a tissue (tumor, healthy cells) in which the targetprotein is detectable. Alternatively, the amounts of protein of a targetgene can be quantified by tissue immunofluorescence using a digitalcamera and suitable software (e.g. Tillvision, Till-photonics, Germany).The technology has frequently been published, and details of stainingand microscopy can therefore be found for example in “DiagnosticImmunohistochemistry” by David J., MD Dabbs ISBN: 0443065667 or“Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: ForLight and Electron Microscopy” ISBN: 0306467704. It should be notedthat, because of the properties of antibodies, different protocols haveto be used (an example is described below) in order to obtain a validresult.

Ordinarily, histologically defined tumor tissues and, as reference,comparable healthy tissues are employed in the IHC. It is moreoverpossible to use as positive and negative controls cell lines in whichthe presence of the target gene is known through RT-PCR analyses. Abackground control must always be included.

Fixed tissue (e.g. fixation with aldehyde-containing substances,formaldehyde, paraformaldehyde or in alcoholic solutions) orshock-frozen tissue pieces with a thickness of 1-10 μm are applied to aglass support. Paraffin-embedded samples are deparaffinated for examplewith xylene. The samples are washed with TBS-T and blocked in serum.This is followed by incubation with the first antibody (dilution: 1:2 to1:2000) for 1-18 hours, with affinity-purified antibodies normally beingused. A washing step is followed by incubation with a second antibodywhich is coupled to an alkaline phosphatase (alternative: for exampleperoxidase), and is directed against the first antibody, for about 30-60minutes. This is followed by color reaction using color substrates whichare converted by the bound enzymes (cf. for example, Shi et al., J.Histochem. Cytochem. 39: 741-748, 1991; Shin et al., Lab. Invest. 64:693-702, 1991). To demonstrate the antibody specificity, the reactioncan be blocked by previous addition of the immunogen.

Immunization

(See also Monoclonal Antibodies: A Practical Approach by PhilipShepherd, Christopher Dean isbn 0-19-963722-9; Antibodies: A LaboratoryManual by Ed Harlow, David Lane ISBN: 0879693142; Using Antibodies: ALaboratory Manual: Portable Protocol NO. by Edward Harlow, David Lane,Ed Harlow ISBN: 0879695447).

The process for preparing antibodies is described briefly below, anddetails can be found in the cited publications. Firstly, animals (e.g.rabbits) are immunized by a first injection of the desired targetprotein. The animal's immune response to the immunogen can be enhancedby a second or third immunization within a defined period (about 2-4weeks after the preceding immunization). Again after various definedperiods (first bleeding after 4 weeks, then about every 2 weeks with atotal of up to 5 samplings), blood is taken from the animals, and animmune serum is obtained therefrom.

The animals are usually immunized by one of four well-establishedmethods, with other methods also being available. It is moreoverpossible to immunize with peptides which are specific for the targetprotein, with the complete protein or with extracellular partialsequences of a protein which can be identified experimentally or viaprediction programs.

-   -   (1) In the first case, peptides (length: 8-12 amino acids)        conjugated to KLH (keyhole limpet hemocyanin) are synthesized by        a standardized in vitro method, and these peptides are used for        the immunization. Usually, 3 immunizations are carried out with        a concentration of 5-1000 μg/immunization. The immunization can        also be carried out as service from service providers.    -   (2) Alternatively, the immunization can be carried out with        recombinant proteins. For this purpose, the cloned DNA of the        target gene is cloned into an expression vector, and the target        protein is synthesized in analogy to the conditions of the        particular manufacturer (e.g. Roche Diagnostics, Invitrogen,        Clontech, Qiagen) for example cell-free in vitro, in bacteria        (e.g. E. coli), in yeast (e.g. S. pombe), in insect cells or in        mammalian cells. After synthesis in one of the systems, the        target protein is purified, the purification in this case        usually taking place by standardized chromatographic methods. It        is also possible in this connection to use for the immunization        proteins which have a molecular anchor as aid for purification        (e.g. His tag, Qiagen; FLAG tag, Roche Diagnostics; Gst fusion        proteins). A large number of protocols is to be found for        example in the “Current Protocols in Molecular Biology”, John        Wiley & Sons Ltd., Wiley Interscience.    -   (3) If a cell line which synthesizes the desired protein        endogenously is available, this cell line can also be used to        produce the specific antiserum. In this case, the immunization        takes place in 1-3 injections in each case with about 1-5×10⁷        cells.    -   (4) The immunization can also take place by injection of DNA        (DNA immunization). For this purpose, the target gene is        initially cloned into an expression vector so that the target        sequence is under the control of a strong eukaryotic promoter        (e.g. CMV promoter). Subsequently, 5-100 μg of DNA are        transferred as immunogen using a “gene gun” into capillary        regions with a strong blood flow in an organism (e.g. mouse,        rabbit). The transferred DNA is taken up by the animal's cells,        the target gene is expressed, and the animal finally develops an        immune response to the target gene (Jung et al., Mol Cells        12:41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21:287-293,        2002).

Quality Control of the Polyclonal Serum or Antibody

Assays based on cell culture with subsequent Western blotting are mostsuitable for demonstrating specificity (various variations are describedfor example in “Current Protocols in Protein Chemistry”, John Wiley &Sons Ltd., Wiley InterScience). For the demonstration, cells aretransfected with a cDNA, which is under the control of a strongeukaryotic promoter (e.g. cytomegalovirus promoter), for the targetprotein. A wide variety of methods (e.g. electroporation, liposome-basedtransfection, calcium phosphate precipitation) are well established fortransfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol.Biol. 75:441-7, 1997). It is also possible alternatively to use celllines which express the target gene endogenously (demonstration bytarget gene-specific RT-PCR). As control, in the ideal case homologousgenes are also transfected in the experiment, in order to be able todemonstrate in the following Western blot the specificity of theanalysed antibody.

In the subsequent Western blot, cells from cell culture or tissuesamples which might contain the target protein are lysed in a 1% SDSsolution, and the proteins are denatured thereby. The lysates arefractionated according to size by electrophoresis on 8-15% denaturingpolyacrylamide gels (contain 1% SDS) (SDS polyacrylamide gelelectrophoresis, SDS-PAGE). The proteins are then transferred by one ofa plurality of blotting methods (e.g. semi-dry electroblot; Biorad) to aspecific membrane (e.g. nitrocellulose, Schleicher & Schüll). Thedesired protein can be visualized on this membrane. For this purpose,the membrane is first incubated with the antibody which recognizes thetarget protein (dilution about 1:20-1:200, depending on the specificityof the antibody) for 60 minutes. After a washing step, the membrane isincubated with a second antibody which is coupled to a marker (e.g.enzymes such as peroxidase or alkaline phosphatase) and which recognizesthe first antibody. It is then possible in a color or chemiluminescentreaction to visualize the target protein on the membrane (e.g. ECL,Amersham Bioscience). An antibody with a high specificity for the targetprotein should in the ideal case recognize only the desired proteinitself.

Various methods are used to confirm the membrane localization of thetarget protein identified in the in silico approach. An important andwell-established method using the antibodies described above isimmuno-fluorescence (IF). Cells of established cell lines which eithersynthesize the target protein (detection of the RNA in an RT-PCR or ofthe protein in a Western blot) or else have been transfected withplasmid DNA are used for this. A wide variety of methods (e.g.electroporation, liposome-based transfection, calcium phosphateprecipitation) are well established for transfection of cell lines withDNA (e.g. Lemoine et al., Methods Mol. Biol. 75:441-7, 1997). Theplasmid transfected into the cells can in the immunofluorescence encodethe unmodified protein or else couple various amino acid markers to thetarget protein. The principal markers are, for example, the fluorescent“green fluorescent protein” (GFP) in its various differentiallyfluorescent forms, short peptide sequences of 6-12 amino acids for whichhigh-affinity and specific antibodies are available, or the short aminoacid sequence Cys-Cys-X-X-Cys-Cys which can bind via its cysteinespecific fluorescent substances (Invitrogen). Cells which synthesize thetarget protein are fixed for example with paraformaldehyde or methanol.The cells can then, if required, be permeabilized by incubation withdetergents (e.g. 0.2% Triton X-100). The cells are then incubated with aprimary antibody which is directed against the target protein or againstone of the coupled markers. After a washing step, the mixture isincubated with a second antibody which is coupled to a fluorescentmarker (e.g. fluorescin, Texas Red, Dako) and which binds to the firstantibody. The cells labeled in this way are then covered with a layer ofglycerol and analysed with the aid of a fluorescence microscopeaccording to the manufacturer's instructions. Specific fluorescenceemissions are achieved in this case by specific excitation depending onthe substances employed. The analysis usually permits reliablelocalization of the target protein, the antibody quality and the targetprotein being confirmed in double stainings to stain in addition to thetarget protein also the coupled amino acid markers or other markerproteins whose localization has already been described in theliterature. GFP and its derivatives represents a special case, beingexcitable directly and themselves fluorescing. The membranepermeability, which can be controlled through the use of detergents,permits demonstration in the immunofluorescence of whether animmunogenic epitope is located inside or outside the cell. Theprediction of the selected proteins can thus be supportedexperimentally. An alternative possibility is to detect extracellulardomains by means of flow cytometry. For this purpose, cells are fixedunder non-permeabilizing conditions (e.g. with PBS/Na azide/2% FCS/5 mMEDTA) and analysed in a flow cytometer in accordance with themanufacturer's instructions. Only extracellular epitopes can berecognized by the antibody to be analysed in this method. A differencefrom immunofluorescence is that it is possible to distinguish betweendead and living cells by use of, for example, propidium iodide or Trypanblue, and thus avoid false-positive results.

Affinity Purification

Purification of the polyclonal sera took place in the case of thepeptide antibodies entirely, or in the case of the antibodies againstrecombinant proteins in part, as service by the contracted companies.For this purpose, in both cases, the appropriate peptide or recombinantprotein was covalently bonded to a matrix, and the latter was, after thecoupling, equilibrated with a native buffer (PBS: phosphate bufferedsaline) and then incubated with the crude serum. After a further PBSwashing step, the antibody was eluted with 100 mM glycine, pH 2.7, andthe eluate was immediately neutralized in 2M TRIS, pH 8. The antibodiespurified in this way could then be employed for specific detection ofthe target proteins both by Western blotting and by immunofluorescence.

Preparation of EGFP Transfectants

For the immunofluorescence microscopy of heterologously expressedtumor-associated antigens, the complete ORF of the antigens was clonedin pEGFP-C1 and pEGFP-N3 vectors (Clontech). CHO and NIH3T3 cellscultivated on slides were transfected with the appropriate plasmidconstructs using Fugene transfection reagent (Roche) in accordance withthe manufacturer's instructions and, after 12-24 h, analysed byimmunofluorescence microscopy.

Example 1 Identification of GPR35 as Diagnostic and Therapeutic CancerTarget

GPR35 (SEQ ID NO:1) and its translation product (SEQ ID NO:9) have beendescribed as putative G protein-coupled receptor. The sequence ispublished in Genbank under accession No. AF089087. This transcript codesfor a protein of 309 amino acids with a molecular weight of 34 kDa. Itwas predicted that GPR35 belongs to the superfamily of G protein-coupledreceptors with 7 transmembrane domains (O'Dowd et al., Genomics47:310-13, 1998). In order to confirm the predicted localization ofGPR35 in the cell, the protein was fused to eGFP as reporter moleculeand, after transfection of the appropriate plasmid, expressedheterologously in 293 cells. The localization was then analysed in afluorescence microscope. It was confirmed according to the inventionthat GPR35 is an integral transmembrane molecule (FIG. 17).Investigation to date on human GPR35 (see, inter alia, Horikawa Y, OdaN, Cox N J, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner T H,Mashima H, Schwarz P E, del Bosque-Plata L, Horikawa Y, Oda Y, YoshiuchiI, Colilla S, Polonsky K S, Wei S, Concannon P, Iwasaki N, Schulze J,Baier L J, Bogardus C, Groop L, Boerwinkle E, Hanis C L, Bell G I NatGenet. 2000 October; 26(2):163-75) suggested that GPR35 is activated inmany healthy tissues. The reading frame of the gene comprises a singleexon. According to the invention, a gene-specific primer pair (SEQ IDNO:20, 21) for GPR35 was used in RT-PCR analyses to amplify cDNA in thecolon and in colon carcinoma (13/26). By contrast, no significantexpression is detectable in other normal tissues. Because of theparticular fact that GPR35 consists of a single exon, genomic DNAimpurities cannot be detected with intron-spanning primers. In order topreclude genomic contamination of the RNA samples, therefore, all RNAswere treated with DNAse. GPR35 transcripts were detected according tothe invention only in the colon, in the rectum, in the testis and incolon carcinomas using DNA-free RNA.

TABLE 1 GPR35 expression in normal tissues Normal tissue ExpressionBrain − Cerebellum − Myocardium − Skeletal muscle − Rectum ++ Stomach −Colon ++ Pancreas − Kidney − Testis − Thymus − Mammary glands − Ovary −Uterus n.d. Skin − Lung − Thyroid − Lymph nodes − Spleen − PBMC −Adrenal − Esophagus − Small intestine + Prostate − (nd = not determined)

The selective and high expression of GPR35 transcripts in normal colonictissue and in colon carcinoma biopsies (FIG. 1) was not previously knownand can be utilized according to the invention for molecular diagnosticmethods such as RT-PCR for detecting disseminating tumor cells in theserum and bone marrow and for detecting metastases in other tissues.Quantitative RT-PCR with specific primers (SEQ ID NO:88 and 89) alsoconfirms that GPR35 is a highly selective intestine-specificdifferentiation antigen which is also contained in intestinal tumors andin intestinal tumor metastases. In some intestinal tumors, it is in factoverexpressed by one log compared with normal intestine (FIG. 18).Antibodies were produced by immunizing rabbits for detecting GPR35protein. The following peptides were used to propagate these antibodies:

GSSDLTWPPAIKLGC (AA 9–23) SEQ ID NO:90 DRYVAVRHPLRARGLR (AA 112–127):SEQ ID NO:91 VAPRAKAHKSQDSLC (C terminus): SEQ ID NO:92 CFRSTRHNFNSMR(extracell. domain 2) SEQ ID NO:93

Stainings with these antibodies for example in a Western blot confirmthe expression in tumors. All 4 extracellular domains of GPR35 (positionof the predicted extracellular domains in the sequence of SEQ ID NO:9 AA1-22 (SEQ ID NO:94); AA 81-94 (SEQ ID NO:95); AA 156-176 (SEQ ID NO:96);AA 280-309 (SEQ ID NO:97)) can be used according to the invention astarget structures of monoclonal antibodies. These antibodies bindspecifically to the cell surface of tumor cells and can be used both fordiagnostic and for therapeutic methods. Overexpression of GPR35 intumors provides additional support for such a use. In addition, thesequences coding for proteins can be used according to the invention asvaccine (RNA, DNA, peptide, protein) for inducing tumor-specific immuneresponses (T-cell and B-cell-mediated immune responses). In addition, ithas surprisingly been found that a further start codon exists 5′ infront of the generally known start codon and expresses an N-terminallyextended protein.

It has thus been found according to the invention that GPR35, a proteinwhich was previously described as expressed ubiquitously, istumor-associated overexpressed, selectively in gastrointestinal tumors,especially in tumors of the colon. GPR35 is therefore suitable inparticular as molecular target structure for the diagnosis and treatmentof these tumors. Investigation to date of human GPR35, cf., for example,Horikawa Y, Oda N, Cox N J, Li X, Orho-Melander M, Hara M, Hinokio Y,Lindner T H, Mashima H, Schwarz P E, del Bosque-Plata L, Horikawa Y, OdaY, Yoshiuchi I, Colilla S, Polonsky K S, Wei S, Concannon P, Iwasaki N,Schulze J, Baier L J, Bogardus C, Groop L, Boerwinkle E, Hanis C L, BellG I Nat Genet. 2000 October;26(2):163-75 suggested that GPR35 isactivated in many healthy tissues. By contrast, the investigationsaccording to the invention showed that GPR35 is surprisingly notsignificantly detectable in most normal tissues and, in contrastthereto, is highly activated in primary and metastatic colon tumors. Inaddition, besides the described GPR35 sequence, according to theinvention a novel translation variant which makes use of an alternativestart codon has been found (SEQ ID NO:10).

GPR35 is a member of the group of G-coupled receptors (GPCR), a verylarge protein family whose structure and function has been very wellinvestigated. GPCR are outstandingly suitable as target structures forthe development of pharmaceutically active substances, because themethods necessary therefor (e.g. receptor expression, purification,ligand screening, mutagenizing, functional inhibition, selection ofagonistic and antagonistic ligands, radiolabeling of ligands) is verywell developed and described in detail, cf., for example, “GProtein-Coupled Receptors” by Tatsuya Haga, Gabriel Berstein and GabrielBernstein ISBN: 0849333849 and in “Identification and Expression ofG-Protein Coupled Receptors Receptor Biochemistry and Methodology” byKevin R. Lynch ASIN: 0471183105. Realization according to the inventionthat GPR35 is undetectable in most healthy tissues but undergoestumor-associated expression on the cell surface, enables it to be usedas tumor-associated target structure for example for pharmaceuticallyactive ligands, especially in conjugation for example with radioactivemolecules as pharmaceutical substances. It is possible in a particularembodiment to use radiolabeled ligands which bind to GPR35 for detectingtumor cells or for treating colon tumors in vivo.

Example 2 Identification of GUCY2C in Hepatic and Ovarian Tumors andNovel GUCY2C Splice Variants as Diagnostic and Therapeutic CancerTargets

Guanylate cyclase 2 C (SEQ ID NO:2; translation product: SEQ ID NO:11)—atype I transmembrane protein—belongs to the family of natriureticpeptide receptors. The sequence is published in Genbank under theaccession number NM_(—)004963. Binding of the peptides guanylin anduroguanylin or else heat-stable enterotoxins (STa) increases theintracellular cGMP concentration, thus inducing signal transductionprocesses inside the cell.

Recent investigations indicate that expression of GUCY2C also extends toextraintestinal regions such as, for example, primary and metastaticadenocarcinomas of the stomach and of the esophagus (Park et al., CancerEpidemiol Biomarkers Prev. 11: 739-44, 2002). A splice variant of GUCYCwhich is found both in normal and transformed tissue of the intestinecomprises a 142 bp deletion in exon 1, thus preventing translation of aGUCY2C-like product (Pearlman et al., Dig. Dis. Sci. 45:298-05, 2000).The only splice variant described to date leads to no translationproduct.

The aim according to the invention was to identify tumor-associatedsplice variants for GUCY2C which can be utilized both for diagnosis andfor therapy.

RT-PCR investigations with a GUCY2C-specific primer pair (SEQ ID NO:22,23, 98, 99) show pronounced expression of GUCY2C transcripts in normalcolon and stomach, and weak expression in liver, testis, ovary, thymus,spleen, brain and lung (tab. 2, FIG. 19). Expression in colon andstomach was at least 50 times higher than in all other normal tissues.Marked GUCY2C transcript levels were detected in colon carcinoma andstomach carcinoma (tab. 2). These results were specified by aquantitative PCR analysis and showed pronounced GUCY2C expression innormal colon, ileum, and in almost all colon carcinoma samplesinvestigated (FIG. 2, 19B). A massive overexpression was detectable insome colon carcinoma samples. In addition, expression is found in 7/10stomach tumors. We also surprisingly found that the gene is activated inmany other previously undescribed tumors, inter alia ovarian, breast,liver and prostate tumors (FIG. 19B, tab. 2).

TABLE 2 GUC2C expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain + Colon carcinoma +++ CerebellumPancreatic − Myocardium carcinoma Skeletal − Esophageal − musclecarcinoma Myocardium Stomach carcinoma +++ Stomach +++ Bronchial − Colon+++ carcinoma Pancreas − Mammary carcinoma −+ Kidney − Ovariancarcinoma + Liver + Endometrial carci Testis ++ ENT tumors Thymus +Renal cell Breast − carcinoma Ovary + Prostate + Uterus + carcinoma SkinLiver carcinoma + Lung + Thyroid Lymph nodes − Spleen + PBMC − Prostate−

The following primer pairs were used to detect splice variants incolonic tissue and colon carcinoma tissue:

GUCY2C-118s/GUCY2C-498as (SEQ ID NO: 24, 29); GUCY2C-621s/GUCY2C-1140as(SEQ ID NO: 25, 30); GUCY2C-1450s/GUCY2C-1790as (SEQ ID NO: 26, 31);GUCY2C-1993s/GUCY2C-2366as (SEQ ID NO: 27, 32);GUCY2C-2717s/GUCY2C-3200as (SEQ ID NO: 28, 33);GUCY2C-118s/GUCY2C-1140as (SEQ ID NO: 24, 30); GUCY2C-621s/GUCY2C-1790as(SEQ ID NO: 25, 31); GUCY2C-1450s/GUCY2C-2366as (SEQ ID NO: 26, 32);GUCY2C-1993s/GUCY2C-3200as (SEQ ID NO: 27, 33).

On investigation of splice variants in colon carcinoma tissue, threepreviously unknown forms were identified according to the invention.

-   -   a) A deletion of exon 3 (SEQ ID NO:3) which leads to a variant        of GUCY2C which is only 111 amino acids long and in which the        asparagine at position 111 is replaced by a proline.    -   b) A deletion of exon 6 (SEQ ID NO:4) which results in an        expression product 258 amino acids long. This would generate a        C-terminal neoepitope comprising 13 amino acids.    -   c) A variant in which the nucleotides at positions 1606-1614,        and the corresponding amino acids L(536), L(537) and Q(538), are        deleted (SEQ ID NO:5).

The splice variants according to the invention with deletionsrespectively in exon 3 and exon 6 (SEQ ID NO:3, 4) are distinguished inparticular by the translation products (SEQ ID NO:12, 13) having notransmembrane domain. The result in the case of exon 6 deletion is aC-terminal neoepitope of 13 amino acids which shows no homologywhatsoever with previously known proteins. This neoepitope is thuspredestined to be a target structure for immunotherapy. The splicevariant of the invention with base deletions at positions 1606-1614 (SEQID NO:5) and its translation product (SEQ ID NO:14) likewise comprises aneoepitope. Antibodies for detecting GUCY2C protein were produced byimmunizing rabbits. The following peptides were used to propagate theseantibodies:

SEQ ID NO:100: HNGSYEISVLMMGNS (AA 31-45)

SEQ ID NO:101: NLPTPPTVENQQRLA (AA 1009-1023)

Such antibodies can in principle be used for diagnostic and therapeuticpurposes.

In particular, the extracellular domain of GUCY2C (position of thepredicted extracellular domain from the sequence of SEQ ID NO:11: AA454-1073 (SEQ ID NO:102)) can be used according to the invention astarget structure of monoclonal antibodies. However, the structuralprediction is somewhat ambiguous and not yet verified experimentally, sothat an alternative membrane orientation is also conceivable. In thiscase, amino acids 1-431 would be outside the cell and be suitable asstarting point for monoclonal antibodies. These antibodies bindspecifically to the cell surface of tumor cells and can be used both fordiagnostic and for therapeutic methods. Overexpression of GUCY2C,especially in the colon tumors, provides additional support for such ause. Sequences coding for proteins can moreover be used according to theinvention as vaccine (RNA, DNA, peptides, protein) for inducingtumor-specific immune responses (T-cell- and B-cell-mediated immuneresponses).

It is moreover possible in accordance with the cellular function of theGUCY2C molecule to develop according to the invention substances,especially small molecules, which modulate the function of the enzyme ontumor cells. The product of the enzymic reaction, cGMP, is a knowncellular signal molecule with a wide variety of functions (Tremblay etal. Mol Cell Biochem 230, 31).

Example 3 Identification of SCGB3A2 as Diagnostic and therapeutic CancerTarget

SCGB3A2 (SEQ ID NO:6) (translation product: SEQ ID NO:15) belongs to thesecretoglobin gene family. The sequence is published in GenBank underaccession number NM_(—)054023. SCGB3A2 (UGRP1) is a homodimericsecretory protein with a size of 17 kDa, which is expressed exclusivelyin the lung and in the spiracles (Niimi et al., Am J Hum Genet70:718-25, 2002). RT PCR investigations with a primer pair (SEQ IDNO:37, 38) confirmed selective expression in normal lung tissue. Lung-and trachea-specific genes, e.g. for surfactant proteins, are highlydownregulated in malignant tumors during dedifferentiation and arenormally undetectable in lung tumors. It was surprisingly found thatSCGB3A2 is active in primary and metastatic lung tumors. Theinvestigations according to the invention showed that SCGB3A2 isstrongly and frequently expressed in bronchial carcinomas (FIG. 4). Allthe other 23 normal tissues tested, apart from lung and trachea, show noexpression (cf. FIG. 20).

This was additionally confirmed in a specific quantitative RT-PCR (SEQID NO:103, 104) (FIG. 20) which additionally shows overexpression by atleast one log in more than 50% of bronchial carcinomas.

The selective and high expression of SCGB3A2 in normal lung tissue andin lung carcinoma biopsies can be used according to the invention formolecular diagnostic methods such as RT-PCR for detecting disseminatingtumor cells in blood and bone marrow, sputum, bronchial aspirate orlavage and for detecting metastases in other tissues, e.g. in locallymph nodes. In the healthy lung, SCGB3A2 is secreted by specializedcells exclusively into the bronchi. Accordingly, it is not to beexpected that SCGB3A2 protein will be detectable in body fluids outsidethe respiratory tract in healthy individuals. By contrast, in particularmetastatic tumor cells secrete their protein products directly into thebloodstream. One aspect of the invention therefore relates to detectionof SCGB3A2 products in serum or plasma of patients via a specificantibody assay as diagnostic finding for lung tumors.

Antibodies for detecting SCGB3A2 protein were produced by immunizingrabbits. The following peptides were used to propagate these antibodies:

LINKVPLPVDKLAPL: SEQ ID NO:105 SEAVKKLLEALSHLV: SEQ ID NO:106

An SCGB3A2-specific reaction was detectable in immunofluorescence (FIG.21). As expected for a secreted protein, the distribution of SCGB3A2 inthe cell was assignable to the endoplasmic reticulum and secretiongranules (FIG. 21A). To check the specificity, the cells weretransfected in parallel with a plasmid that synthesizes an SCGB3A2-GFPfusion protein. Protein detection took place in this case via theautofluorescent GFP (green fluorescent protein) (FIG. 21B).Superimposition of the two fluorescence diagrams shows unambiguouslythat the immune serum specifically recognizes SCGB3A2 protein (FIG.21C). Such antibodies can be used according to the invention for examplein the form of immunoassays for diagnostic and therapeutic purposes.

Example 4 Identification of Claudin-18A1 and Claudin-18A2 SpliceVariants as Diagnostic and Therapeutic Cancer Targets

The claudin-18 gene codes for a surface membrane molecule having 4transmembrane domains and intracellular N terminus and C terminus. Niimiand colleagues (Mol. Cell. Biol. 21:7380-90, 2001) describe two splicevariants of the murine and human claudin-18 which have been described asexpressed selectively in lung tissue (claudin-18A1) and in stomachtissue (claudin-18A2), respectively. These variants differ in the Nterminus (FIG. 22).

It was investigated according to the invention how far the splicevariants claudin-18A2 (SEQ ID NO:7) and claudin-18A1 (SEQ ID NO:117),and their respective translation products (SEQ ID NO:16 and 118), can beused as markers or therapeutic target structures for tumors. Aquantitative PCR able to distinguish between the two variants wasestablished by selecting A1-specific (SEQ ID NO:109 & 110) andA2-specific (SEQ ID NO:107 & 108) primer pairs. The A2 splice variantwas additionally tested with a second primer pair in a conventional PCR(SEQ ID NO:39 & 40). The A1 variant is described to be active only innormal lung. However, it was surprisingly found according to theinvention that the A1 variant is also active in the gastric mucosa.Stomach and lung are the only normal tissues showing significantactivation. All other normal tissues are negative for claudin-A1. Oninvestigating tumors, it was surprisingly found that claudin-A1 ishighly activated in a large number of tumor tissues. Particularly strongexpression is to be found in stomach tumors, lung tumors, pancreaticcarcinomas, esophageal carcinomas (FIG. 23), ENT tumors and prostatecarcinomas. The claudin-A1 expression levels in ENT, prostate,pancreatic and esophageal tumors are 100-10,000 higher than the levelsin the corresponding normal tissues. The oligonucleotides used toinvestigate the claudin-A2 splice variant specifically enable thistranscript to be amplified (SEQ ID NO:39 & 40 and 107 & 108).Investigation revealed that the A2 splice variant is expressed in noneof the more than 20 normal tissues investigated apart from gastricmucosa and to a small extent also testis tissue. We have found that theA2 variant is also, like the A1 variant, activated in many tumors(depicted by way of example in FIG. 24). These include stomach tumors(8/10), pancreatic tumors (6/6), esophageal carcinomas (5/10) and livercarcinomas. Although no activation of claudin-18A2 is detectable inhealthy lung, it was surprisingly found that some lung tumors expressthe A2.1 splice variant.

TABLE 3A Expression of claudin-18A2 in normal and tumor tissues Normaltissue Expression Tumor type Expression Brain − Colon − Cerebellum −carcinoma Myocardium − Pancreatic ++ Skeletal − carcinoma muscleEsophageal ++ Endometrium − carcinoma Stomach +++ Gastric +++ Colon −carcinoma Pancreas − Bronchial ++ Kidney − carcinoma Liver − Breast −Testis + carcinoma Thymus − Ovarian − Breast − carcinoma Ovary −Endometrial n.i. Uterus − carcinoma Skin − ENT tumors ++ Lung − Renalcell − Thyroid − carcinoma Lymph nodes − Prostate − Spleen − carcinomaPBMC − Esophagus −

TABLE 3B Expression of claudin-18A1 in normal and tumor tissues Normaltissue Expression Tumor type Expression Brain − Colon − Cerebellum −carcinoma Myocardium − Pancreatic ++ Skeletal − carcinoma muscleEsophageal ++ Endometrium − carcinoma Stomach +++ Gastric +++ Colon −carcinoma Pancreas − Bronchial ++ Kidney − carcinoma Liver − Breast +Testis + carcinoma Thymus − Ovarian n.i. Breast − carcinoma Ovary −Endometrial n.i. Uterus − carcinoma Skin − ENT tumors ++ Lung +++ Renalcell − Thyroid − carcinoma Lymph nodes − Prostate ++ Spleen − carcinomaPBMC − Esophagus −

Conventional PCR as independent control investigation also confirmed theresults of the quantitative PCR. The oligonucleotides (SEQ ID NO:39, 40)used for this permit specific amplification of the A2 splice variant. Itwas shown according to the invention that 8/10 gastric carcinomas andhalf of the tested pancreatic carcinomas showed strong expression ofthis splice variant (FIG. 5). By contrast, expression is not detectablein other tissues by conventional PCR. In particular, there is noexpression in lung, liver, blood, lymph nodes, breast tissue and kidneytissue (tab. 3).

The splice variants thus represent according to the invention highlyspecific molecular markers for tumors of the upper gastrointestinaltract as well as lung tumors, ENT tumors, prostate carcinomas andmetastases thereof. These molecular markers can be used according to theinvention for detecting tumor cells. Detection of the tumors is possibleaccording to the invention with the oligonucleotides described (SEQ IDNO:39, 40, 107-110). Particularly suitable oligonucleotides are primerpairs of which at least one binds under stringent conditions to asegment of the transcript which is 180 base pairs long and is specificfor one (SEQ ID NO:8) or the other splice variant (SEQ ID NO:119).

In order to confirm these data at the protein level, claudin-specificantibodies and immune sera were generated by immunizing animals. Theplasma membrane localization of claudin-18 and the protein topology wasconfirmed by analysis of the transmembrane domains with bioinformatictools (TMHMM, TMPRED) and immunofluorescence investigations of cellswhich expressed claudin-18 fusion proteins tagged with enhanced GFP.Claudin-18 has two extracellular domains. The N-terminal extracellulardomain differs in sequence in the two splice variants (SEQ ID NO:111 forA1 and SEQ ID NO:112 for A2). The C-terminal extracellular domain isidentical for both variants (SEQ ID NO:137) To date, no antibodies whichbind to the extracellular domains of claudin-18 have yet been described.According to the invention, peptide epitopes which are locatedextracellularly and are specific for variant A1 or A2 or occur in bothvariants were selected for the immunization. Both variants of claudin-18have no conventional glycosylation motifs and the glycosylation of theprotein was therefore not to be expected. Nevertheless, account wastaken in the selection of the epitopes that epitopes which compriseasparagine, serine, threonine are potentially glycosylated in rare caseseven without conventional glycosylation sites. Glycosylation of anepitope may prevent the binding of an antibody specific for thisepitope. Inter alia, epitopes were selected according to the inventionso that the antibodies generated thereby permit the glycosylation statusof the antigen to be distinguished. The following peptides, inter alia,were selected for producing antibodies for the immunization:

SEQ ID NO:17: DQWSTQDLYN (N-terminal extracellular domain, A2-specific,binding independent of glycosylation)

SEQ ID NO:18: NNPVTAVFNYQ (N-terminal extracellular domain, A2-specific,binding mainly to unglycosylated form, N37)

SEQ ID NO:113: STQDLYNNPVTAVF (N-terminal extracellular domain,A2-specific, binding only to non-glycosylated form, N37)

SEQ ID NO:114: DMWSTQDLYDNP (N-terminal extracellular domain,A1-specific)

SEQ ID NO:115: CRPYFTILGLPA (N-terminal extracellular domain, mainlyspecific for A1)

SEQ ID NO:116: TNFWMSTANMYTG (C-terminal extracellular domain,recognizes both A1 and A2).

The data for the A2-specific antibody produced by immunization with SEQID NO:17 are shown by way of example. The specific antibody can beutilized under various fixation conditions for immunofluorescenceinvestigations. With comparative stainings of RT-PCR-positive andnegative cell lines, in an amount which is readily detectable, thecorresponding protein can be specifically detected in the gastriccarcinoma cell lines typed as positive (FIG. 25). The endogenous proteinis membrane-located and forms relatively large focal aggregates on themembrane. This antibody was additionally employed for protein detectionin Western blotting. As expected, protein is detected only in stomachand in no other normal tissue, not even lung (FIG. 29). The comparativestaining of stomach tumors and adjacent normal stomach tissue frompatients surprisingly revealed that claudin-18 A2 has a smaller massweight in all stomach tumors in which this protein is detected (FIG. 30,left). It was found according to the invention in a series ofexperiments that a band also appears at this level when lysate of normalstomach tissue is treated with the deglycosylating agent PNGase F (FIG.30, right). Whereas exclusively the glycosylated form of the A2 variantis detectable in all normal stomach tissues, A2 is detectable as such inmore than 60% of the investigated gastric carcinomas, in particularexclusively in the deglycosylated form. Although the A2 variant ofclaudin-18 is not detected in normal lung even at the protein level, itis to be found in bronchial carcinomas, as also previously in thequantitative RT-PCR. Once again, only the deglycosylated variant ispresent (FIG. 31). Antibodies which recognize the extracellular domainof the claudin-18-A2 splice variant have been produced according to theinvention. In addition, antibodies which selectively recognize theN-terminal domain of the claudin-18-A1 splice variant (FIG. 28) andantibodies which bind to both variants in the region of the C-terminalextracellular domain (FIG. 27) have been produced. It is possibleaccording to the invention to use such antibodies for diagnosticpurposes, e.g. immunohistology (FIG. 32), but also for therapeuticpurposes as explained above. A further important aspect relates todifferentially glycosylated domains of claudin-18. Antibodies whichexclusively bind to non-glycosylated epitopes have been producedaccording to the invention. Claudin-18 itself is a highly selectivedifferentiating antigen for stomach tissue (A2) and for the lung andstomach (A1). Since it is evidently affected by changes in theglycosylation machinery in tumors, a particular, deglycosylated, variantof A2 is produced in tumors. This can be utilized diagnostically andtherapeutically. Immune sera such as the one described here (againstpeptide of SEQ ID NO:17) can be utilized diagnostically for example inWestern blotting. Antibodies which are entirely unable to bind to theglycosylated epitope as obtained for example by immunization withpeptide of SEQ ID NO:113 (FIG. 26), can distinguish tumor tissue fromnormal tissue in the binding. It is possible in particular to employsuch antibodies therapeutically because they are highly selective. Theproduced antibodies can be used directly also for producing chimeric orhumanized recombinant antibodies. This can also take place directly withantibodies obtained from rabbits (concerning this, see J Biol Chem. 2000May 5;275(18):13668-76 by Rader C, Ritter G, Nathan S, Elia M, Gout I,Jungbluth A A, Cohen L S, Welt S, Old L J, Barbas C F 3rd. “The rabbitantibody repertoire as a novel source for the generation of therapeutichuman antibodies”). For this purpose, lymphocytes from the immunizedanimals were preserved. The amino acids 1-47 (SEQ ID NO:19 and 120) alsorepresent particularly good epitopes for immunotherapeutic methods suchas vaccines and the adoptive transfer of antigen-specific T lymphocytes.

Example 5 Identification of SLC13A1 as Diagnostic and Therapeutic CancerTarget

SLC13A1 belongs to the family of sodium sulfate cotransporters. Thehuman gene is, in contrast to the mouse homolog of this gene,selectively expressed in the kidney (Lee et al., Genomics 70:354-63).SLC13A1 codes for a protein of 595 amino acids and comprises 13 putativetransmembrane domains. Alternative splicing results in 4 differenttranscripts (SEQ ID NO:41-44) and its corresponding translation products(SEQ ID NO:45-48). It was investigated whether SLC13A1 can be used asmarker for kidney tumors. Oligonucleotides (SEQ ID NO:49, 50) whichenable specific amplification of SLC13A1 were used for this purpose.

TABLE 4 Expression of SLC13A1 in normal and tumor tissues Normal tissueExpression Tumor type Expression Brain − Colon nd Cerebellum ndcarcinoma Myocardium nd Pancreatic nd Skeletal nd carcinoma muscleEsophageal nd Myocardium − carcinoma Stomach − Gastric nd Colon −carcinoma Pancreas nd Bronchial nd Kidney +++ carcinoma Liver − Breastnd Testis + carcinoma Thymus − Ovarian nd Breast − carcinoma Ovary −Endometrial nd Uterus nd carcinoma Skin nd ENT tumors nd Lung − Renalcell +++ Thyroid − carcinoma Lymph nodes − Prostate nd Spleen −carcinoma PBMC − Sigmoid − Esophagus −

RT-PCR investigations with an SLC13A1-specific primer pair (SEQ IDNO:49, 50) confirmed virtually selective expression in the kidney, andshowed according to the invention a high expression in virtually all(7/8) investigated renal cell carcinoma biopsies (tab. 4, FIG. 6).Quantitative RT-PCR with specific primers (SEQ ID NO:121, 122) alsoconfirmed these data (FIG. 34). Weak signals were detectable in thefollowing normal tissues: colon, stomach, testis, breast, liver andbrain. Expression in renal carcinomas was, however, at least 100 timeshigher than in all other normal tissues.

In order to analyse the subcellular localization of SLC13A1 in the cell,the protein was fused to eGFP as reporter molecule and, aftertransfection of the appropriate plasmid, expressed heterologously in 293cells. The localization was then analysed under the fluorescencemicroscope. Our data impressively confirmed that SLC13A1 is an integraltransmembrane molecule (FIG. 35).

Antibodies for detecting the SLC13A1 protein were produced by immunizingrabbits. The peptides of SEQ ID NO:123 and 124 were used for propagatingthese antibodies. Such antibodies can in principle be used fordiagnostic and therapeutic purposes.

The SLC13A1 protein has 13 transmembrane domains and 7 extracellularregions. These extracellular domains of SLC13A1 in particular can beused according to the invention as target structures for monoclonalantibodies. SLC13A1 is involved as channel protein in the transport ofions. The extracellular domains of SLC13A1 in the healthy kidney aredirected polarically in the direction of the urinary tract (luminally).However, high molecular weight monoclonal antibodies employedtherapeutically are not excreted into the urinary tract, so that nobinding to SLC13A1 takes place in the healthy kidney. By contrast, thepolarity of SLC13A1 is abolished in tumor cells, and the protein isavailable for antibody targeting directly via the bloodstream. Thepronounced expression and high incidence of SLC13A1 in renal cellcarcinomas make this protein according to the invention a highlyinteresting diagnostic and therapeutic marker. This includes accordingto the invention the detection of disseminated tumor cells in serum,bone marrow, urine, and detection of metastases in other organs by meansof RT-PCR. It is additionally possible to use the extracellular domainsof SLC13A1 according to the invention as target structure forimmunodiagnosis and therapy by means of monoclonal antibodies. SLC13A1can moreover be employed according to the invention as vaccine (RNA,DNA, protein, peptides) for inducing tumor-specific immune responses (Tand B cell-mediated immune responses). This includes according to theinvention also the development of so-called small compounds whichmodulate the biological activity of SLC13A1 and can be employed for thetherapy of renal tumors.

Example 6 Identification of CLCA1 as diagnostic and Therapeutic CancerTarget

CLCA1 (SEQ ID NO:51; translation product: SEQ ID NO:60) belongs to thefamily of Ca⁺⁺-activated Cl⁻ channels. The sequence is published inGenbank under the accession No. NM_(—)001285. CLCA1 is exclusivelyexpressed in the intestinal crypt epithelium and in the goblet cells(Gruber et al., Genomics 54:200-14, 1998). It was investigated whetherCLCA1 can be used as marker for colonic and gastric carcinoma.Oligonucleotides (SEQ ID NO:67, 68) which enable specific amplificationof CLCA1 were used for this purpose. RT-PCR investigations with thisprimer set confirmed selective expression in the colon, and showedaccording to the invention high expression in (3/7) investigated colonicand (1/3) investigated gastric carcinoma samples (FIG. 7). The othernormal tissues showed no or only very weak expression. This wasadditionally confirmed with a specific quantitative RT-PCR (SEQ IDNO:125, 126), in which case no expression could be detected in thenormal tissues analyzed (FIG. 36). Of the tumor samples investigated inthis experiment, 6/12 colonic carcinoma samples and 5/10 gastriccarcinoma samples were positive for CLCA1. Overall, expression of thegene in tumors appears to be dysregulated. Besides samples with verystrong expression, CLCA1 was markedly downregulated in other samples.

The protein is predicted to have 4 transmembrane domains with a total of2 extracellular regions. These extracellular domains of CLCA1 inparticular can be used according to the invention as target structuresfor monoclonal antibodies.

The pronounced expression and high incidence of CLCA1 in gastric andcolonic carcinomas make this protein according to the invention aninteresting diagnostic and therapeutic marker. This includes accordingto the invention the detection of disseminated tumor cells in serum,bone marrow, urine, and detection of metastases in other organs by meansof RT-PCR. It is additionally possible to use the extracellular domainsof CLCA1 according to the invention as target structure forimmunodiagnosis and therapy by means of monoclonal antibodies. CLCA1 canmoreover be employed according to the invention as vaccine (RNA, DNA,protein, peptides) for inducing tumor-specific immune responses (T and Bcell-mediated immune responses). This includes according to theinvention also the development of so-called small compounds whichmodulate the biological activity as transport proteins of CLCA1 and canbe employed for the therapy of gastrointestinal tumors.

Example 7 Identification of FLJ21477 as Diagnostic and TherapeuticCancer Target

FLJ21477 (SEQ ID NO:52) and its predicted translation product (SEQ IDNO:61) was published as hypothetical protein in Genbank under theaccession No. NM_(—)025153. It is an integral membrane protein havingATPase activity and 4 transmembrane domains, which is accordinglysuitable for therapy with specific antibodies. RT-PCR investigationswith FLJ21477-specific primers (SEQ ID NO:69, 70) showed selectiveexpression in the colon, and additionally various levels of expressionin (7/12) investigated colonic carcinoma samples (FIG. 8). The othernormal tissues showed no expression. This was confirmed additionally bya specific quantitative RT-PCR (SEQ ID NO:127, 128). FLJ21477-specificexpression was detectable both in colon (FIG. 37A) and in 11/12 ofcolonic carcinomas. Besides the expression in colon tissue, expressionwas additionally detectable in stomach tissue. In addition, under theconditions of the quantitative RT-PCR, the expression detectable inbrain, thymus and esophagus was distinctly weaker compared with colonand stomach (FIG. 37A). It was moreover additionally possible to detectFLJ21477-specific expression in the following tumor samples: stomach,pancreas, esophagus and liver.

The protein is predicted to have 4 transmembrane domains with a total of2 extracellular regions. These extracellular domains of FLJ21477 inparticular can be used according to the invention as target structuresfor monoclonal antibodies.

The expression and the high incidence of FLJ21477 for gastric andcolonic carcinomas make this protein according to the invention avaluable diagnostic and therapeutic marker. This includes according tothe invention the detection of disseminated tumor cells in serum, bonemarrow, urine, and the detection of metastases in other organs by meansof RT-PCR. In addition, the extracellular domains of FLJ21477 can beused according to the invention as target structure for immunodiagnosisand therapy by means of monoclonal antibodies. In addition, FLJ21477 canbe employed according to the invention as vaccine (RNA, DNA, protein,peptides) for inducing tumor-specific immune responses (T and Bcell-mediated immune responses).

Example 8 Identification of FLJ20694 as Diagnostic and TherapeuticCancer Target

FLJ20694 (SEQ ID NO:53) and its translation product (SEQ ID NO:62) werepublished as hypothetical protein in Genbank under accession No.NM_(—)017928. This protein is an integral transmembrane molecule(transmembrane domain AA 33-54), very probably with thioredoxinfunction. RT-PCR investigations with FLJ20694-specific primers (SEQ IDNO:71, 72) showed selective expression in the colon, and additionallyvarious levels of expression in (5/9) investigated colonic carcinomasamples (FIG. 9). The other normal tissues showed no expression. Thiswas additionally confirmed by a specific quantitative RT-PCR (SEQ IDNO:129, 130) (FIG. 38). FLJ29694 expression was undetectable in anyother normal tissue apart from colon and stomach (not analysed in thefirst experiment).

The protein is predicted to have one transmembrane domain with anextracellular region. These extracellular domains of FLJ20694 inparticular can be used according to the invention as target structuresfor monoclonal antibodies.

In addition, FLJ20694 can be employed according to the invention asvaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immuneresponses (T and B cell-mediated immune responses). This includesaccording to the invention also the development of so-called smallcompounds which modulate the biological activity of FLJ20694 and can beemployed for the therapy of gastrointestinal tumors.

Example 9 Identification of Von Ebner's Protein (c20orf114) asDiagnostic and Therapeutic Cancer Target

von Ebner's protein (SEQ ID NO:54) and its translation product (SEQ IDNO:63) were published as Plunc-related protein of the upper airways andof the nasopharyngeal epithelium in Genbank under the accession No.AF364078. It was investigated according to the invention whether vonEbner's protein can be used as marker of lung carcinoma.Oligonucleotides (SEQ ID NO:73, 74) which enable specific amplificationof Ebner's protein were used for this purpose. RT-PCR investigationswith this primer set showed selective expression in the lung and in(5/10) investigated lung carcinoma samples (FIG. 10). In the group ofnormal tissues there was also expression in the stomach. The othernormal tissues showed no expression.

Example 10 Identification of Plunc as Diagnostic and Therapeutic CancerTarget

Plunc (SEQ ID NO:55) and its translation product (SEQ ID NO:64) werepublished in Genbank under the accession No. NM_(—)016583. Human Plunccodes for a protein of 256 amino acids and shows 72% homology with themurine Plunc protein (Bingle and Bingle, Biochem Biophys Acta1493:363-7, 2000). Expression of Plunc is confined to the trachea, theupper airways, nasopharyngeal epithelium and salivary gland.

It was investigated according to the invention whether Plunc can be usedas marker of lung carcinoma. Oligonucleotides (SEQ ID NO:75, 76) whichenable specific amplification of Plunc were used for this purpose.

RT-PCR investigations with this primer set showed selective expressionin the thymus, in the lung and in (6/10) investigated lung carcinomasamples (FIG. 11). Other normal tissues showed no expression.

Example 11 Identification of SLC26A9 as Diagnostic and TherapeuticCancer Target

SLC26A9 (SEQ ID NO:56) and its translation product (SEQ ID NO:65) werepublished in Genbank under the accession No. NM_(—)134325. SLC26A9belongs to the family of anion exchangers. Expression of SLC26A9 isconfined to the bronchiolar and alveolar epithelium of the lung (Lohi etal., J Biol Chem 277:14246-54, 2002).

It was investigated whether SLC26A9 can be used as marker of lungcarcinoma. Oligonucleotides (SEQ ID NO:77, 78) which enable specificamplification of SLC26A9 were used for this purpose. RT-PCRinvestigations with SLC26A9-specific primers (SEQ ID NO:77, 78) showedselective expression in the lung and in all (13/13) investigated lungcarcinoma samples (FIG. 12). The other normal tissues showed noexpression, with the exception of the thyroid. It was possible inquantitative RT-PCR experiments with the primers of SEQ ID NO:131 and132 firstly to confirm these results, and to obtain additionalinformation. It was possible in pooled samples of 4-5 tumor tissues todetect high expression levels for SLC26A9-specific RNA in lung, colon,pancreas and stomach tumors. SLC26A9 is member of a family oftransmembrane anion transporters. In the healthy lung, the protein isluminally directed in the direction of the airways and thus not directlyavailable to IgG antibodies from the blood. By contrast, the polarity ofthe protein is abolished in tumors. It is therefore possible accordingto the invention to address SLC26A9 as therapeutic target usingmonoclonal antibodies in the defined tumors, inter alia lung tumors,gastric carcinomas, pancreatic carcinomas. The pronounced, highexpression and high incidence of SLC26A9 for lung, stomach, pancreaticand esophageal carcinomas make this protein according to the inventionan excellent diagnostic and therapeutic marker. This includes accordingto the invention the detection of disseminated tumor cells in serum,bone marrow and urine, and detection of metastases in other organs bymeans of RT-PCR. In addition, the extracellular domains of SLC26A9 canbe used according to the invention as target structure forimmunodiagnosis and therapy by means of monoclonal antibodies. It isadditionally possible to employ SLC26A9 according to the invention asvaccine (RNA, DNA, protein, peptides) for inducing tumor-specific immuneresponses (T and B cell-mediated immune responses). This includesaccording to the invention also the development of so-called smallcompounds which modulate the biological activity of SLC26A9 and can beemployed for the therapy of lung tumors and gastrointestinal tumors.

Example 12 Identification of THC1005163 as Diagnostic and TherapeuticCancer Target

THC1005163 (SEQ ID NO:57) is a gene fragment from the TIGR gene index.The gene is defined only in the 3′ region, while an ORF is lacking.RT-PCR investigations took place with a THC1005163-specific primer (SEQID NO:79) and an oligo dT₁₈ primer which had a specific tag of 21specific bases at the 5′ end. This tag was examined using databasesearch programs for homology with known sequences. This specific primerwas initially employed in the cDNA synthesis in order to precludegenomic DNA contaminations. RT-PCR investigations with this primer setshowed expression in the stomach, ovary, lung and in (5/9) lungcarcinoma biopsies (FIG. 13). Other normal tissues showed no expression.

Example 13 Identification of LOC134288 as mediated Diagnostic andTherapeutic Cancer Target

LOC134288 (SEQ ID NO:58) and its predicted translation product (SEQ IDNO:66) were published in Genbank under accession No. XM_(—)059703.

It was investigated according to the invention whether LOC134288 can beused as marker of renal cell carcinoma. Oligonucleotides (SEQ ID NO:80,81) which enable specific amplification of LOC134288 were used for thispurpose. RT-PCR investigations showed selective expression in the kidneyand in (5/8) investigated renal cell carcinoma biopsies (FIG. 14).

Example 14 Identification of THC943866 as Diagnostic and TherapeuticCancer Target

THC 943866 (SEQ ID NO:59) is a gene fragment from the TIGR gene index.It was investigated whether THC943866 can be used as marker of renalcell carcinoma. Oligonucleotides (SEQ ID NO:82, 83) which enablespecific amplification of THC943866 were used for this purpose.

RT-PCR investigations with THC943866-specific primers (SEQ ID NO:82, 83)showed selective expression in the kidney and in (4/8) investigatedrenal cell carcinoma biopsies (FIG. 15).

Example 15 Identification of FLJ21458 as Diagnostic and TherapeuticCancer Target

FLJ21458 (SEQ ID NO:84) and its predicted translation product (SEQ IDNO:85) were published in Genbank under the accession No. NM_(—)034850.Sequence analyses revealed that the protein represents a new member ofthe butyrophillin family. Structural analyses revealed that itrepresents a type 1 transmembrane protein with an extracellularimmunoglobulin domain. Oligonucleotides (SEQ ID NO:86, 87) which enablespecific amplification of FLJ21458 were used for investigatingexpression. RT-PCR investigations with FLJ21458-specific primers (SEQ IDNO:86, 87) showed selective expression in colon and in (7/10)investigated colonic carcinoma biopsies (FIG. 16, tab. 5). QuantitativeRT-PCR with specific primers (SEQ ID NO:133, 134) confirmed thisselective expression profile (FIG. 39). It was additionally possible inthe experiment to detect FLJ21458 gastrointestinal-specifically in thecolon, and in stomach, in the rectum and cecum and in testis. 7/11 colonmetastasis samples were also positive in the quantitative PCR.FLJ21458-specific expression was extended to other tumors, and aprotein-specific expression was detectable in stomach, pancreas andliver tumors (tab. 5). Antibodies for detecting FLJ21458 protein wereproduced by immunizing rabbits. The following peptides were used topropagate these antibodies:

QWQVFGPDKPVQAL: SEQ ID NO:135 AKWKGPQGQDLSTDS: SEQ ID NO:136

An FLJ21458-specific reaction was detectable in immuno-fluorescence(FIG. 40). To check the specificity of the antibodies, 293 cells weretransfected with a plasmid that codes for an FLJ21458-GFP fusionprotein. Specificity was demonstrated on the one hand by colocalizationinvestigations using the FLJ21458-specific antibody, and on the otherhand via the auto-fluorescent GFP. Superimposition of the twofluorescent diagrams showed unambiguously that the immune serumspecifically recognises FLJ21458 protein (FIG. 40 a). Owing to theoverexpression of the protein, the resultant cell staining was diffuseand did not allow unambiguous protein localization. For this reason, afurther immunofluorescence experiment was carried out with the stomachtumor-specific cell line Snu16 which expresses FLJ21458 endogenously(FIG. 41B). The cells were stained with the FLJ21458-specific antiserumand with another antibody which recognizes the membrane proteinE-cadherin. The FLJ21458-specific antibody stains the cell membranes atleast weakly and is thus evidence that FLF21458 is localized in the cellmembrane.

Bioinformatic investigations showed that the protein encoded by FLJ21458represents a cell surface molecule and has an immunoglobulinsupermolecule domain. Selective expression of this surface moleculemakes it a good target for developing diagnostic methods for thedetection of tumor cells and therapeutic methods for the elimination oftumor cells.

The pronounced expression and high incidence of FLJ21458 for gastric andcolonic carcinomas make this protein according to the invention a highlyinteresting diagnostic and therapeutic marker. This includes accordingto the invention the detection of disseminated tumor cells in serum,bone marrow and urine, and the detection of metastases in other organsby means of RT-PCR. It is additionally possible to employ theextracellular domains of FLJ21458 according to the invention as targetstructure for immuno-diagnosis and therapy by means of monoclonalantibodies. It is additionally possible to employ FLJ21458 according tothe invention as vaccine (RNA, DNA, protein, peptides) for inducingtumor-specific immune responses (T and B cell-mediated immuneresponses). This includes according to the invention also thedevelopment of so-called small compounds which modulate the biologicalactivity of FLJ21458 and can be employed for the therapy ofgastrointestinal tumors.

TABLE 5 FLJ21458 expression in normal and tumor tissues Normal tissueExpression Tumor type Expression Brain − Colonic 7/10 Cerebellum −carcinoma Myocardium nd Pancreatic 5/6  Skeletal − carcinoma muscleEsophageal nd Myocardium − carcinoma Stomach ++ Gastric 8/10 Colon +++carcinoma Pancreas − Bronchial nd Kidney − carcinoma Liver − Breast ndTestis ++ carcinoma Thymus nd Ovarian nd Breast nd carcinoma Ovary −Endometrial nd Uterus − carcinoma Skin − ENT tumors nd Lung − Renal cellnd Thyroid nd carcinoma Lymph nodes − Prostate nd Spleen − carcinomaPBMC − Colonic 7/11 Adrenal nd metastases Esophagus − Liver 5/8  Small −carcinoma intestine Prostate −

1. A method of diagnosing lung, pancreatic, or esophageal cancer,comprising detecting expression of a tumor-associated antigen in abiological sample, wherein the tumor-associated antigen is selected fromthe group consisting of: (i) the polypeptide of SEQ ID NO: 16; and (ii)the polypeptide encoded by the nucleic acid of SEQ ID NO: 7, whereindetection of the tumor-associated antigen in a biological sampleisolated from a patient in an amount greater than an amount of thetumor-associated antigen in a normal biological sample indicates thepresence of lung, pancreatic, or esophageal cancer.
 2. The method ofclaim 1, in which the detection comprises: (i) contacting the biologicalsample with an antibody which binds specifically to the tumor-associatedantigen; and (ii) detecting a complex formed between the antibody andthe tumor-associated antigen.
 3. The method of claim 2, wherein theantibody is labeled with a detectable marker.
 4. The method of claim 3,wherein the detectable marker is a radioactive marker or an enzymaticmarker.
 5. The method of claim 1, wherein the biological samplecomprises body fluid or body tissue.